The ability of Lactobacillus sake to use various carbon sources was investigated. For this purpose we developed a chemically defined medium allowing growth of L. sake and some related lactobacilli. This medium was used to determine growth rates on various carbohydrates and some nutritional requirements of L. sake. Mutants resistant to 2-deoxy-d-glucose (a nonmetabolizable glucose analog) were isolated. One mutant unable to grow on mannose and one mutant deficient in growth on mannose, fructose, and sucrose were studied by determining growth characteristics and carbohydrate uptake and phosphorylation rates. We show here that sucrose, fructose, mannose, N-acetylglucosamine, and glucose are transported and phosphorylated by the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). The PTS permease specific for mannose, enzyme II(supMan), was shown to be responsible for mannose, glucose, and N-acetylglucosamine transport. A second, non-PTS system, which was responsible for glucose transport, was demonstrated. Subsequent glucose metabolism involved an ATP-dependent phosphorylation. Ribose and gluconate were transported by PTS-independent permeases.