We describe the multifactorial origins of catalysis by the twister ribozyme. We provide evidence that the adenine immediately 3′ to the scissile phosphate (A1) acts as a general acid. Substitution of ring nitrogen atoms indicates that very unusually the N3 of A1 is the proton donor to the oxyanion leaving group. A1 is accommodated in a specific binding pocket that raises its pKa toward neutrality, juxtaposes its N3 with the O5′ to be protonated, and helps create the in-line trajectory required for nucleophilic attack. A1 performs general acid catalysis while G33 acts as a general base. A 100-fold stereospecific phosphorothioate effect at the scissile phosphate is consistent with a significant stabilization of the transition state by the ribozyme, and functional group substitution at G33 indicates that its exocyclic N2 interacts directly with the scissile phosphate. A model of the ribozyme active site is proposed that accommodates these catalytic strategies.