Classical techniques for the isolation of C. tetani appear even in the most recent textbooks, because few investigations have been made in this area in recent years. A reappraisal of the isolation methods of C. tetani, including the use of GAM (Gifu Anaerobic Medium), which is commonly used but hitherto unexplored for this purpose, appeared to be important in view of some conflicting results reported in the literature. The following cultural conditions were reappraised: i) Media for enrichment culture and the duration of incubation, ii) media for isolation of C. tetani from the enrichment culture employing agar slant media, and iii) the recovery rate of C. tetani from the enrichment medium employing the optimal agar slant medium. As samples from which C. tatani is isolated usually contain other bacteria, we studied the isolation of C. tetani mixed experimentally with other bacteria, employing different media under several conditions. The following results were obtained: 1) The analysis of the growth curves of C. tetani, cultured simultaneously with E. coli, Streptococcus sp., group G, S. aureus and C. perfringens, revealed that GAM broth is the best medium as compared with the liver, cooked meat and thioglycollate broth media recommended by others. 2) GAM, blood and heart infusion agar slant media were equally useful for the isolation of C. tetani from the mixed culture with E. coli. There was no indication that GAM agar medium inhibited the swarming of C. tatani. The swarming colonies of C. tetani were more easily detected when the GAM agar slant was used than when the blood agar slant medium was used. 3) Glucose in the heart infusion agar medium inhibited the swarming of C. tetani at a concentration of 1%, and both growth and swarming of C. tetani were inhibited when the concentration of glucose was raised to 2%. In view of this finding, Zeissler's medium, which contains 2% glucose is not recommended for this purpose. C. tetani failed to be recovered from samples mixed with Streptococcus sp., group G or with C. perfringens, even when GAM agar slant medium was used, when the dose of C. tetani added to the broth was not sufficiently large. C. tetani must reach a certain level of density to be recovered from mixed cultures, and by means of plural, not a singular agar slant media. This is particularly important when clinical, soil and stool samples which are contaminated with an unknown number of bacterial species are subjected to isolation.