Two bacteriological methods, one to demonstrate phosphatase production and the other to demonstrate aesculin hydrolysis, were tested on sterol-requiring Mycoplasma and sterol-nonrequiring Acholeplasma strains to determine their utility in differentiating species. The phosphatase test, which gave weak and variable reactions with most of the sterolnonrequiring strains, was judged ineffectual for differentiating these strains. The aesculin test distinguished all Mycoplasma strains and Acholeplasma granularum, both of which gave negative reactions, from most Acholeplasma laidlawii strains and from Acholeplasma axanthum, which gave positive reactions.