An extracellular lipase of Streptomyces rimosus R6-554W was isolated from the culture filtrate by column chromatography using diethylaminoethyl-cellulose, carboxymethyl-cellulose, hydroxylapatite, Mono S (fast protein liquid chromatography), and Sephadex G-75. It was shown to be a monomeric, basic protein (M-r = 27 500, pI = 8.45), active toward triolein and p-nitrophenyl esters, with preference for those with medium size (C-8-C-12) acyl chain length. Interfacial activation was observed with p-nitrophenyl butyrate as substrate. The lipase was most active at 50-60 degrees C and in alkaline conditions around pH 9-10, with p-nitrophenyl palmitate as substrate. It showed high stability at a broad pH range of 4-10 and was fairly thermostable. Dithiothreitol moderately inactivated the enzyme. Phenylmethylsulfonyl fluoride partly inhibited lipase only when added during the hydrolytic reaction.