Aims: A lipase-encoding gene (lip/A) from a psychrotrophic strain of Pseudomonas fluorescens C9 has previously been characterized. It was also shown that when this gene was insertionally-inactivated, lipase activity was retained, suggesting that a second lipase may be present in this strain. The aim of this study was to determine whether this was the case. Methods and Results: Using molecular cloning, chromosomal mutagenesis and enzymatic analysis, the presence of a second lipase-encoding gene (lipB) has been confirmed. The molecular weights of the putative products of lipA and lipB are 33 and 64.5 kDa, respectively, and their sequences are quite dissimilar (< 10% sequence identity). The lipB gene encodes a secreted lipase and is solely responsible for the 'lipolytic phenotype' of Ps. fluorescens C9. Expression of the lipA gene can be detected when expressed using an expression vector, but activity was only detected intracellularly in Ps. fluorescens C9, and not in the culture medium. Conclusions: Pseudomonas fluorescens C9 contains two dissimilar lipases. One (LipB) is secreted and responsible for the lipolytic phenotype; the evidence suggests that the other (LipA) could be intracellular, but it could be secreted and not detectable. Significance and Impact of the Study: Bacteria mag; contain more than one lipase activity. Ascribing phenotypes to particular enzymes therefore requires mutational analysis. The notion of an intracellular lipase activity is novel, and, if further substantiated, begs the question as to its normal substrate and physiological role.