The polyamide layer technique for the chromatographic separation of dimethylaminonaphthalene sulphonyl amino acids has been adapted to the qualitative analysis of amino acids in media before and after the growth of micro-organisms. The method has been used to study the amino acids metabolized by cultures of proteolytic clostridia growing in a medium consisting of an acid hydrolysate of casein as a source of amino acids and small amounts of yeast extract and trypticase as sources of growth factors. The chromatograms of the media after growth showed which amino acids were used and which new amino acids were produced. Clostridium botulinum type F (proteolytic), C. ghoni, C. mangenoti and C. putrificum were found to reduce proline to 5-aminovaleric acid and to produce 2-aminobutyric acid, properties they shared with C. sporogenes and C. sticklandii. C. botulinum type G and C. subterminale used glycine, lysine, serine, and arginine but in contrast to C. sticklandii they neither reduced proline to 5-aminovaleric acid nor produced 2-aminobutyric acid. Both organisms oxidized phenylalanine, tyrosine and tryptophan to phenylacetic acid, p-hydroxyphenyl acetic acid and indole acetic acid respectively. C. lituseburense and C. scatologenes used serine, threonine and arginine and produced 2-amino butyric acid and ornithine. C. lentoputrescens, C. limosum and C. malenomenatum resembled C. tetanomorphum by using glutamic acid and tyrosine. The chromatograms always showed the physiological group to which an organism belonged and in some cases were characteristic of the species.