Nonreplicative transposition by Tn10/IS10 involves three chemical steps at each transposon end: cleavage of the two strands plus joining of one strand to target DNA. These steps occur within a synaptic complex comprising two transposon ends and monomers of IS10 transposase. We report four transposase mutations that individually abolish each of the three chemical steps without affecting the synaptic complex. We conclude that a single constellation of residues, the "active site," directly catalyzes each of the three steps. Analyses of reactions containing mixtures of wild-type and catalysis-defective transposases indicate that a single transposase monomer at each end catalyzes the cleavage of two strands and that strand transfer is carried out by the same monomers that previously catalyzed cleavage. These and other data suggest that one active site unit carries out all three reactions in succession at one transposon end.