A thermostable lipase from Pseudomonas cepacia has been purified to homogeneity as judged by SDS-PAGE and isoelectric focusing. The purification included treatment of the culture supernatant with acrinol, hydrophobic interaction chromatography, and gel filtration. The enzyme was a monomeric protein with M_r of 36,500 and pI of 5.1. The optimal pH at 50°C and optimal temperature at pH 6.5 were 5.5–6.5 and 55–60°C, respectively, when olive oil was used as the substrate. Simple triglycerides of short and middle chain fatty acids (C ≤ 12) were the preferred substrates over those of long chain fatty acids. The enzyme cleaved all the ester bonds of triolein, with some preference for the 1,3-ester bonds. The enzyme retained all its activity even after incubation at 75°C (pH 6.5) for 30 min. Further, the activity was not impaired during 21 h storage at pH 6.5 in 40% water-miscible solvents including methanol, ethanol, acetone, acetonitrile, dimethylfonnamide, dimethylsulfox-ide, and dioxane. The addition of dimethylsulfoxide or acetone to the assay mixture in the range of 0–35% stimulated the enzyme, whereas benzene or n-hexane had an inhibitory effect. These properties together with the N-terminal amino acid sequence confirmed that the enzyme differs from the known Pseudomonas sp. lipases.