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Mega Satan is located behind the golden gate at the starting room of The Chest and the Dark Room. To unlock Mega Satan, the following criteria must be fulfilled:
??? or The Lamb must have been defeated in an earlier run.
In subsequent runs, Angel Statues can be bombed to fight an Angel boss. When killed, they will drop either Key Piece 1 or Key Piece 2.
Collecting both key pieces will form the fully completed key, which is automatically used upon entering The Chest or the Dark Room on the golden gate.
Alternatively, Dad's Key can be used to unlock the Golden Key Door in lieu of the key pieces. Dad's Key is unlocked in the basement by collecting both key pieces in an earlier run.
Passing through this Golden Key door and stepping onto the pentagram will initiate the Mega Satan fight.