Buffering capacity and membrane H+ conductance were examined in three gram-positive bacteria, Staphylococcus aureus, Bacillus subtilis, and Bacillus alcalophilus. An acid pulse technique was used to measure both parameters. The buffering capacity and membrane H+ conductance of B. alcalophilus are influenced by the pH of the medium and the culture conditions. Suspensions of B. alcalophilus cells from both H. A. medium and l-malate medium cultures grown at pH 10.5 exhibited higher values for these parameters than cells grown at pH 8.5. B. alcalophilus grown aerobically had a lower buffering capacity and a lower membrane conductance for protons than the neutrophilic bacteria S. aureus and B. subtilis. Fermenting cells exhibited significantly higher values for both variables than respiring cells.