Not counting DLC characters, there are 155 playable characters in the game. For example, Spider-Man has his 'spider senses' that allow him to locate hidden bricks required for advancing the gameplay in levels, and the 'big-figs' like the Hulk and Abomination can destroy or manipulate objects in ways even different than characters with normal super strength. LEGO Marvel Super Heroes includes some new powers and abilities as well, however. For example, heat-related characters can destroy gold bricks, rocket-using characters can destroy silver bricks, super heroes that have the power of flight in the comic books can fly in the game, and characters with super strength can destroy or manipulate LEGO objects that normal characters cannot. LEGO Marvel Superheroes also carries over a large number of features that were included in LEGO Batman 2: DC Super Heroes. In addition, there are a variety of side-levels to embark on.

The player progresses through a number of levels following a linear plot, but can return to the levels later on in free play mode, using any of the characters they unlocked up until that point. Like a majority of the LEGO games created by TT Games, players take control of characters that are modeled like actual LEGO parts (but with far more mobility) and are divided into groups according to certain skills. Nick Fury calls upon the Avengers ( Iron Man, Captain America, Thor, Hulk, Black Widow, and Hawkeye), the Fantastic Four ( Mister Fantastic, Invisible Woman, Human Torch, and Thing), Spider-Man, the X-Men ( Professor X, Wolverine, Jean Grey, Cyclops, Storm, Colossus, Iceman, Beast and Gambit) and various other Marvel heroes, to deal with the threat against the world.

With the ray Doctor Doom wishes to defeat the Devourer of Worlds and show the people of Earth he will guide them all to peace. Loki and Doctor Doom assemble a team of villains (including Venom, Doctor Octopus, Magneto and his Brotherhood of Mutants, Sandman, Arnim Zola, Green Goblin and the Abomination) to collect the bricks to build 'Doctor Doom's Doom Ray of Doom'. His surfboard is destroyed and becomes cosmic bricks, that are spread all over the world. He is chased by Iron Man and is attacked by Doctor Doom. The Silver Surfer comes to Earth to herald Galactus' arrival.

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