Spores of Clostridium botulinum type 62A were germinated in a chemically defined medium (8 mm l-cysteine, 11.9 mm sodium bicarbonate, 4.4 mm sodium thioglycolate; buffered with 100 mm TES, pH 7.0). The rate and extent of germination were increased when an aqueous spore suspension was heated sublethally (80 C, 60 min) before addition to the germination medium. Neither sublethal nor lethal doses of gamma radiation had any marked effect on subsequent germination. Maximum germination (>90% in 2 hr) in the defined medium occurred in the pH range of 6.5 to 7.5, at 30 to 37 C, with an l-cysteine level of 8 mm. Increasing l-cysteine to 32 mm increased the rate (over that with 8 mm l-cysteine) but not the extent of germination. The rate and extent of germination increased with NaHCO3 addition to 8.3 mm, but increasing levels to 11.9 mm had no further effect. For maximum germination, 2.2 mm sodium thioglycolate was required and higher levels (to 8.8 mm) had no further enhancing or inhibitory effect. Under optimal conditions for germination, 97% of the spores had become heat sensitive; 98% had become sensitive to radiation; 88 and 91% had become phase dark and stainable, respectively, and the spore suspension had lost 46% of its initial optical density by 2 hr. Loss of heat resistance preceded loss of radiation resistance, acquisition of stainability, and phase darkening by about 12 min.