The lipase, phosphatase, and aryl sulfatase activities of 387 mycobacterial cultures of various species of 4 groups according to Ragnion were studied with the aim of differentiation of these bacteria. Phosphatase activity was shown by M. fortuitum, M. phlei, M. marinum, M. bovis, M. tuberculosis, M. avium, M. intracellulare, and Group III nonphotochromogenic Mycobacteria (according to Ragnion); M. smegmatis and M. thamnopheos negatively reacted to phosphatase; and the cultures of Group II Mycobacteria showed heterogeneous phosphatase activity. M. phlei, M. smegmatis, M. fortuitum, M. scrofulaceum and M. gordonae showed marked lipase activities; M. bovis, M. marinum, M. thamnopheo, M. avium, M. intracellulare, nonphotochromogenous Mycobacteria of Group III, M. terrae and M. gastri were negative. In the rest mycobacterial cultures the lipase activity varied. Only M. fortuitum and some cultures of M. scrofulaceum, M. marinum, M. avium, M. intracellulare and Group II Mycobacteria showed aryl sulfatase activity.