Tn5-induced insertion mutations were generated in the Pseudomonas syringae pv. tomato genome by mating this plant pathogen with an Escherichia coli strain carrying the suicide plasmid vector for Tn5, pGS9. Kmr transconjugants occurred at frequencies ranging from 2 × 10−7 to 9 × 10−6; approximately 5.5% of these transconjugants were also Cmr, indicating the presence of additional pGS9 DNA sequences. Approximately 1% of the Kmr Cms mutants were auxotrophic. Southern blot analysis revealed that the Tn5 element had inserted into one unique site on the chromosome for each Kmr Cms transconjugant examined. Physical and genetic tests of Tn5-induced auxotrophs showed that Tn5 mutations in P. syringae pv. tomato were very stable and that secondary transposition of Tn5 or its insertion sequence IS50 was a rare event. Nine of 920 Kmr Cms transconjugants screened on tomato seedlings either were avirulent or produced very mild symptoms. Each of the virulence mutants was the result of a unique single-site Tn5 insertion. Five mutants also failed to induce a hypersensitivity reaction on tobacco.