If Mario Kart or F-Zero are not going to headline E3- and since they've already been unveiled (and in F-Zero's case, played by the press in Japan), I too doubt they actually will- Nintendo will have to have either a franchise of equal or greater repute or something all-new. Since there's no way for me to speak knowledgeably about anything all-new, lets go down the usual suspects.
Any GameBoy game: I don't think a GameBoy game can headline E3 for Nintendo. Even though it's Nintendo's cash cow, they need to pimp something on the console to get its rep up, and they'd be better off using Mario Kart to headline rather than a Game Boy game.
Mario: could be, as Miyamoto has stated it's already in development. But I'm not convinced just yet. I expect something on video, though. But a short video clip probably isn't good enough to headline a show.
Zelda: Another possibility, but considering the Wind Waker will only have been out less than 2 weeks in Europe when E3 comes around, If anything Zelda is shown, I don't expect anything more than a short video clip, again, not highlight material
Metroid: I don't think Prime 2 is far enough im develop to have something playable. Video at most.
Kid Icarus: As geeked as I'd be to see a new Kid Icarus game, I gave up hope after Icarus wasn't a playable character in Smash Bros. Melee and just had a stupid-looking trophy (I still contend he'd be perfect for the game). Still, he's not big enough to headline a show, unless they do something really dramatic with it. I'd like to see a third person action game like Zelda/Star Fox Adventures, but with the character capable of flight! That would be freaking great. But I doubt that could headline.
Earthbound: It's already been unveiled (in a small way), and I HIGHLY doubt Nintendo would use a game that stars a little round boy with a magic yo-yo and a waddling sidekick with a huge schnoz to headline the biggest show of the year when they're trying to shake their 'kiddy' image.
Donkey Kong: This would work nicely. If it exists.
Donkey Kong Racing: I fell in love with the video clip shown so long ago, but with Rare over with Microsoft, I think this is a dead game. Anyway, with Diddy Kong and Donkey Kong both in Mario Kart, and there might be even more DKC characters as hidden playables, I'm not sure there's a point of this game even existing anymore, even without Rare.
Pikmin 2: Same boat as Mario Kart
Something by Silicon Knights: Hmmm...Nintendo might want to use something by these folk to emphasize their commitment to shaking the kiddy image, but I don't think so.
Ice Climbers: Uh.......no. How many people actually knew of the Ice Climbers before Smash Bros. Melee? Exactly
Smash Bros. 3: Not a headliner, but I would love to see it. I probably wouldn't have a GameCube if it wasn't for Melee.
This is my pick, barring anything brand new and unexpected:
Star Fox Armada
Nintendo said they're going to focus on third parties at this show. Armada is made by Namco. Star Fox has the 'star power' to headline a show. Not quite as much as Metroid, Mario, or Zelda, for sure, but it's still one of Nintendo's best franchises. Plus, peopple have been clamoring for a return to the Star Fox/Star Fox 64 style game for a while now, so the anticipation for this game is high. Plus, we haven't seen any of Armada yet, save for a poster,so Nintendo can unveil it in a big way. The big problem with Armada is that it isn't due until next year (at least I think), so it may not be appropriate for a headliner, but I'm sure Microsoft will use Halo 2 as at least a co-headliner (along with something from Rare I think, like Perfect Dark 2) and that's not due until next year, so who knows.