A method for the formation of the trimethylsilyl (TMS) derivatives of the wholecell hydrolysate of bacteria was developed. The TMS derivatives were examined by gas-liquid chromatography. TMS profiles of various bacteria at the genus and species level were compared. Differences in TMS profiles of Listeria, Neisseria, and Clostridium were significant as were differences between the TMS profiles of C. perfringens and C. sporogenes. Two types of C. perfringens, two serotypes of L. monocytogenes, and one culture of C. sporogenes and N. meningitidis were studied. The possible application of TMS profiles as an aid in differentiating closely related organisms which are troublesome to separate by conventional means is discussed.