The structural gene (lipA) coding for the extracellular lipase of Pseudomonas aeruginosa PAO1 has been cloned on plasmid pSW118. Nucleotide sequence analysis revealed a gene of 936 bp. lipA codes for a proenzyme of 311 amino acids including a leader sequence of 26 amino acids. The mature protein was predicted to have a M(r) of 30134, an isoelectric point of 5.6, and a consensus sequence (IGHSHGG) typical of lipases. Furthermore it is highly homologous (greater than 60%) to other lipases from various pseudomonads. The lipA gene failed to hybridize detectably with genomic DNA from other Pseudomonas species except P. alcaligenes, even under relaxed stringency. Located 220 bp downstream of the lipA gene, is an open reading frame (ORF2, lipH) which encodes a hydrophilic protein (283 amino acids; M(r) 33587) that shows some homology to the limA gene product of P. cepacia. In complementation tests of lipase-defective mutants, lipH was shown to be necessary for expression of active extracellular lipase in P. aeruginosa PAO1.