Although 47 strains of Clostridium difficile and Clostridium sporogenes were studied by gas chromatography. Acidic and neutral volatile products, formed after 96 h of incubation in glucose-containing peptone yeast-extract medium, were chromatographed. All strains produced appreciable amounts of fatty acids, which were tentatively identified by gas chromatographic retention data. Chromatograms obtained when analysing diethyl ether extracts of culture media of all 47 strains were virtually identical. However, analysis of the broth media by automatic head-space gas chromatography, employing a glass capillary column, gave chromatographic patterns which differentiated the two Clostridium species studied. C. sporogenes was characterized by chromatographic patterns containing a dominant peak of isovaleric acid. Strains of this species produced larger amounts of early eluting compounds and much smaller amounts of butyric and valeric acid than did strains of C. difficile. Automatic head-space gas chromatography provides an efficient means for differentiation of C. difficile and C. sporogenes. This gas chromatographic technique is easier and more rapidly performed than analysis of either extracts of culture media.