Experiments were undertaken on the biosynthetic pathway to coronatine in Pseudomonas syringae pv. glycinea, using mutants blocked in coronatine synthesis, wild type strains and radioactive labelling with L-[U-14C] isoleucine and 14C-coronamic acid. Evaluation of the kinetics of coronatine appearance in the medium revealed an initial exponential phase with a maximum rate of 0.86 pmol hr-1 culture-1 after 3.1 days growth. Beyond this the rate declined to a steady rate of 0.20 pmol hr-1 culture-1. With P. syringae 4185,8.6% of L-[U-14C] isoleucine added at three days growth was incorporated into coronatine during a 90 min exposure period. At the same time there was negligible incorporation (0.04%) to coronamic acid. Two mutants blocked in coronatine synthesis were shown to produce coronamic acid by isolation and purification of this product from culture supernatants. L-[U-14C]Isoleucine was added to one of these mutants at four days growth and 1.9% of the radioactivity was incorporated into coronamic acid, these data showed that coronamic acid is a distinct biosynthetic entity. However, the coronatine-producing strain from which these mutants were derived, P. syringae 4180, showed only 0.08% incorporation into a coronamic acid fraction, although culture supernatants after five days growth did reveal a trace of coronamic acid as detected by ninhydrin after electrophoresis and chromatography on thin layer cellulose plates. When a three-day culture of P. syringae 4185 was fed coronamic acid prior to 14C-isoleucine, a 3.7-fold reduction in the incorporation of label into coronatine occurred; instead the radioactivity was diverted to extracelluar coronamic acid which showed 7% incorporation. This result demonstrates that coronamic acid is a biosynthetic intermediate of coronatine. Pseudomonas syringae 4185 very efficiently utilized 14C-coronamic acid, giving 94% incorporation into coronatine after 90 min exposure in a four-day culture, showing the specific utilization of this compound. Other experiments were undertaken to evaluate the possible involvement of coronafacoylisoleucines in the biosynthesis of coronatine, by use of a carmutant blocked in coronafacic acid synthesis, with the conclusion that this was not an operative pathway to coronatine.