The role of F1F0-ATPase in Streptococcus mutans GS5 was investigated by isolating a mutant (NTS1) defective in enzyme activity by homologous recombination with a plasmid encoding the 5' terminal fragment of the F1F0-ATPase beta-subunit gene. The ATPase activity of NTS1 membranes was 49% that of GS5 membranes. The lag phase of the growth curve of NTS1 was longer than that for GS5, and the lag phase of GS5 and NTS1 were prolonged by the addition of ionophore gramicidin D; at stationary phase, the turbidity of the NTS1 culture was less than that of the GS5 culture. These results suggest that S. mutans F1F0-ATPase contributes to the generation of a stoichiometric electrogenic gradient effectively in the lag phase.