A nitrocellulose colony-blot assay was developed to detect enterotoxigenic strains of Clostridium perfringens on an agar medium. To enhance sporulation and enterotoxin production, a number of modifications of the Duncan-Strong (D-S) medium were tested, including the substitution of raffinose for starch and the addition of theobromine, papaverine and various combinations of soil and fecal extracts. Colonies of enterotoxigenic strains were most consistently positive and produced the most intense color reactions on a modified D-S medium containing raffinose, theobromine and 50% (vol/vol) bovine fecal extract. This modified medium stimulated production of detectable enterotoxin by colonies in more than 90% of the enterotoxigenic strains tested. No false-positive reactions were observed. This enzyme-linked, immunosorbent assay (ELISA) was not as effective in the analysis of broth cultures or fecal samples. Our results indicate that the nitrocellulose colony-blot assay will be useful for screening enterotoxigenic strains in epidemiologic studies.