Pseudomonas syringae pv. tomato PT23.2 produces the chlorosis-inducing phytotoxin coronatine. Thirty-eight chlorosis-defective mutants of PT23.2 were previously generated by using the transposon Tn5. Five mutants contained Tn5 insertions in the indigenous plasmid pPT23A; the remaining 33 mutants either were missing pPT23A (29 mutants) or contained deletions in this plasmid (4 mutants). These results suggested that pPT23A was involved in coronatine production in strain PT23.2. This plasmid was introduced into P. syringae pv. syringae PS61, which does not produce coronatine. A bioassay for coronatine suggested that PS61(pPT23A) transconjugants were able to make this phytotoxin. In a chemical analysis, organic acids were isolated from PT23.2, PS61, and the transconjugant PS61(pPT23A); these were derivatized to their methyl esters and analyzed by gas chromatography. The derivatized organic acids extracted from PT23.2 and PS61(pPT23A) contained peaks that corresponded to coronafacic acid, coronafacoylvaline, and coronatine, but these were absent in the extracts from the wild-type strain PS61. The identification of these components was confirmed by combined gas chromatography-mass spectrophotometry. Therefore, the acquisition of pPT23A by PS61 resulted in biosynthesis of coronafacic acid, coronafacoylvaline, and coronatine, clearly demonstrating the involvement of pPT23A in coronatine production in P. syringae pv. tomato.