Lipase activity produced during cultivation of Fusarium oxysporum f. sp. vasinfectum in shake flasks was predominantly extracellular. Only a small amount was found in or on the mycelium. The fungus required peptone for significant lipase production. Addition of trimyristin, olive oil, Span 85, and oleic acid to growing shake-flask cultures, and the addition of olive oil and oleic acid to suspensions of washed mycelium, enhanced the level of lipase activity produced. Synthesis of lipase activity was repressed by glucose and glycerol. It is suggested that catabolite repression of lipase formation in F. oxysporum f. sp. vasinfectum was mainly regulated by transcriptional control. Lipase activity was inhibited by oleic acid and its sodium salt, bur not by glycerol. The highest lipase activity was measured with monoacid triglycerides of middle chain fatty acids (C-8-C-12). Diethyl p-nitrophenyl phosphate reduced the activity toward triolein and tributyrin almost to the same extent and with the same velocity. The lipase activity remained constant during Ih incubation at pH 4.0 to 10.5 and 30 degrees C. It was stable in 25% (v/v) acetone, ethanol and n-propanol and was stimulated remarkably in n-hexane.