As in different microbial species, two pathways for the degradation of glycerol are found in B. subtilis 168. Each pathway includes two enzymes which can catalyze the formation of dihydroxyacetone phosphate from glycerol in vitro. The first pathway includes a glycerol dehydrogenase (gl-D) and a dihydroxyacetone kinase (dha-K). The second pathway includes a glycerol kinase (gl-K) and an α-glycerophosphate dehydrogenase (glp-D). Enzymes of both pathways are repressed in the presence of glucose. Only the enzymes of the second pathway are inducible. The inducer is probably glycerophosphate, utilization of which as a carbon source by B. subtilis is demonstrated. Degradation of glycerol in B. subtilis proceeds through the second pathway. This was demonstrated by the isolation of a mutant (gl−2) impaired in glycerol kinase, and which cannot use glycerol as a carbon source. Another mutant (gl−1)was isolated, which cannot use glycerol as a carbon source. When comparing the activity of the four enzymes, particularly gl-K, no significant differences were observed between the wild strain and the mutant gl−1. This leads one to consider the existence of a glycerol permeation system in B. subtilis. A mutation affecting this system would explain the behavior of the mutant gl−1.