Bacillus brevis strain Nagano and its gramicidin S-negative mutant, BI-7, were compared with respect to germination of their spores produced in several media. Germination initiation occurred in the presence of nutrient broth or L-alanine but not with inosine, glucose, glycerol or fructose; the process was activated by heat. Parental and mutant spores behaved similarly in these experiments. During outgrowth, parental spores remained in this phase of germination much longer than did mutant spores, but only when the parental spores had been harvested from a sporulation medium where significant gramicidin S synthesis had occurred. When parental spores were extracted or treated with an enzyme that hydrolyzes gramicidin S, rapid outgrowth occurred. Adding exogenous gramicidin S or the extract from parental spores to mutant spores lengthened the outgrowth in a dose-dependent manner. The uptake of labeled L-alanine by parental spores was delayed compared to mutant spores in the presence or absence of chloramphenicol. These data suggest a mechanism of action for gramicidin S whereby it interferes in membrane function, such as transport or energy metabolism, in outgrowing spores.