Group I introns possess a single active site that catalyzes the two sequential reactions of self-splicing. An RNA comprising the two domains of the Tetrahymena thermophila group I intron catalytic core retains activity, and the 5.0 angstrom crystal structure of this 247-nucleotide ribozyme is now described. Close packing of the two domains forms a shallow cleft capable of binding the short helix that contains the 5[prime] splice site. The helix that provides the binding site for the guanosine substrate deviates significantly from A-form geometry, providing a tight binding pocket. The binding pockets for both the 5[prime] splice site helix and guanosine are formed and oriented in the absence of these substrates. Thus, this large ribozyme is largely preorganized for catalysis, much like a globular protein enzyme.