Streptococcus mutans DR0001 and a glucose-phosphotransferase (PTS)-defective mutant, DR0001/6, were grown anaerobically in a chemostat with a glucose limitation at dilution rates (D) of 0.04 to 0.6 h(-1) (mean generation time, 17 to 1.2 h). The mutant possessed only 15% of glucose-PTS activity of the wild type and gave cell yields (19%) less than those of the wild type. Glucose-PTS activity in strains DR0001 was maximum at D = 0.1 h(-1) and was adequate to account for transport in the chemostat at all dilution rates except D = 0.6 h(-1), at which it was 80% of the actual glucose uptake activity. The mutant DR0001/6, on the other hand, possessed only sufficient glucose-PTS activity to sustain growth at below D = 0.1 h(-1), indicating the presence of an alternate transport activity. This was confirmed in glycolytic rate experiments with washed cells, which demonstrated that the mutant showed rates 11- to 27-fold higher than that accountable via glucose-PTS activity alone. The wild-type organism contained both a high (K(s), 6.7 to 8.0 muM)- and a low (K(s), 57 to 125 muM)-affinity transport system, whereas the glucose-PTS-defective mutant contained only the low-affinity system (K(s), 62 to 133 muM). The glucose-PTS was shown to be the high-affinity system. Glucose uptake by the mutant was unaffected by 8 mM sodium arsenate, 10 mM azide, and 10 mM dinitrophenol but was completely inhibited by 0.05 mM sodium iodoacetate. Glycolysis in the organism was almost completely inhibited by 0.25 mM N',N' -dicyclohexylcarbodiimide (DCCD), indicating the involvement of an ATPase in glucose uptake. The ionophores carbonylcyanide-m-chlorophenylhydrazone and tetrachlorosali-cylanilide were inhibitory at concentrations of 10 muM, suggesting that a proton gradient was important in the transport process. Higher levels of DCCD and the ionophores were required to inhibit the wild-type organism to the same degree. A mechanism is proposed for the alternative transport system whereby proton motive force is created by the extrusion of protons by the DCCD-sensitive ATPase and glucose is transported down a proton gradient in a symport with protons.