IS10 transposase mediates end pairing, donor cleavage (or excision) and strand transfer reactions in IS10/Tn10 transposition. Details of how this multi-functional protein mediates these various steps have not been determined. We have used a genetic approach to help define structure-function relationships for IS10 transposase. IS10 transposase mutants that failed to form an excised transposon fragment in vivo and conferred a dominant-negative phenotype were isolated. Mutants with these properties were expected to be defective in catalysis and/or steps preceding donor cleavage. A total of seven mutants were identified, the corresponding proteins were purified and their activities assessed in vitro. Mutant EK292 was found to form wild-type levels of paired end complex, an early intermediate in the reaction, but to be defective in both donor cleavage and strand transfer. This phenotype suggests that E292 comprises part of a catalytic region. Mutants RH119, SF120 and RQ296 formed aberrant paired end complexes, indicating a defect in a step(s) preceding donor cleavage. Mutants WR98, IS101, and AV162 were found to promote donor cleavage in vitro but not strand transfer. For IS101 and AV162 the defect in strand transfer may be related to instability of excision complexes. Finally, the observation that IS101, RH119 and AV162 were greatly suppressed by Mn2+ suggests that these mutations have an effect on Mg2+ interactions with transposase.