ABSTRACTA 65 kDa intracellular lipase fromLactobacillus plantarum2739 was purified to homogeneity (482‐fold, specific activity of 251 μmol/mg per min) and characterized. The purification procedure included chromatography with Q‐Sepharose, Sephacryl 200, Phenyl‐Superose and Mono Q. The purified lipase was optimally active at pH 7.5 and 35C; it retained about 40% of the maximum activity at pH 5.0 and 15C. The enzyme was stable at 65C (D65C= 18.6 min) and was irreversibly inactivated at 75C for 2 min. On triglycerides, the highest activity was determined on tributyrin but trilaurin and tripalmitin were hydrolyzed also. The Km on tributyrin was 2.31 mM. β‐Naphthyl esters of fatty acids from C2 to C12 were hydrolyzed with a preference for β‐naphthyl butyrate. After lipolysis, the fatty acid profiles in β‐monoacylglycerols of milk fat showed similarities among porcine pancreatic lipase, rennet paste and lipase fromLb. plantarum2739, but the bacterial enzyme caused a greater hydrolysis of C10 and C12 fatty acids esterified at the Sn‐2 position of glycerol. The lipase was strongly inhibited by 1 mM Nethylmaleimide and iodoacetic acid, by 10 mM Hg2+and Ag+, and was moderately stimulate