Pseudomonas aeruginosa PAO1 mutants affected in acyclic monoterpenes, n-octanol, and acetate assimilation were isolated using transposon mutagenesis. The isocitrate lyase gene (aceA) corresponding to ORF PA2634 of the PAO1 strain genome was identified in one of these mutants. The aceA gene encodes a protein that is 72% identical to the isocitrate lyase (ICL) characterized from Colwellia maris, but is less than 30% identical to their homologues from pseudomonads. The genetic arrangement of aceA suggests that it is a monocistronic gene, and no adjacent related genes were found. The ICL protein was detected as a 60-kDa band in sodium dodecyl sulfate polyacrylamide gel electrophoresis from cultures grown on acetate, but not in glucose-grown PAO1 cultures. Genetic complementation further confirmed that the aceA gene encodes the ICL enzyme. The ICL enzyme activity in crude extracts from cultures of the PAO1 strain was induced by acetate, citronellol and leucine, and repressed by growth on glucose or citrate. These results suggest that ICL is involved in the assimilation of acetate, acyclic monoterpenes of the citronellol family, alkanols, and leucine, in which the final intermediary acetyl-coenzyme A may be channelled to the glyoxylate shunt.