The amount of F1Fo-ATPase in Enterococcus hirae (formerly Streptococcus faecalis) increases when the cytoplasmic pH is lowered below 7.6, and protons are extruded to maintain the cytoplasmic pH at around 7.6. In the present study, we found that the transcriptional activity of the F1Fo-ATPase operon was not regulated by pH. The synthesis of F1 subunits was increased 1.65 +/- 0.12-fold by the acidification of medium from pH 8.0 to pH 5.3. Western-blot analysis showed that there were F1 subunits in the cytoplasm, and the number of alpha plus beta subunits in the cytoplasm was 50% of the total number of the subunits in cells growing at pH 8.0. This decreased to 22% after shifting the medium pH to 5.3, with a concomitant 5.1-fold increase in the level of membrane-bound F1Fo-ATPase. The cytoplasmic F1 subunits were shown to be degraded, and Fo subunits not assembled into the intact F1Fo complex were suggested to be digested. These data suggest that regulation of the enzyme level of F1Fo-ATPase by the intracellular pH takes place mainly at the step of enzyme assembly from its subunits.