Four strains of Mycoplasma pneumoniae, seven strains of M. salivariam, two strains of M. orale, two strains of M. hominis, and one strain of M. fermentons were grown for 6 days in PPLO broth containing horse serum and yeast extract. The cultures were harvested by centrifugation, and the resulting sediments were digested with tetramethylammonium hydroxide. Ethyl alcohol suspensions of the digestion products were analyzed by gas chromatography. All the species except M. pneumoniae produced three distinctive chromatographic peaks. Two of these peaks were reproducible, and M. salivarium, M. orale, M. hominis, and M. fermentons could be differentiated on the basis of the peak height ratios.