We examined H' and HC03-transport mechanisms that are involved in the regulation of intracellular pH of Schwann cells. Primary cultures of Schwann cells were prepared from the sciatic nerves of 1-3-day-old rats. pHi of single cells attached to cover slips was continuously monitored by measuring the absorbance spectra of the pH-sensitive dye dimethylcarboxyfluorescein incorporated intracellularly. The average pHi of neonatal Schwann cells bathed in HEPES mammalian solution was 7.17 * 0.02 (n = 32). In the nominal absence of HC03-, pHi spontaneously recovered from an acute acid load induced by exposing the Schwann cells to 20 mM NH,+ (NH,' prepulse). This pHi recovery from the acute acid load was totally inhibited in the absence of external Na' or in the presence of 1 mM amiloride. In both cases, the pHi recovery was readily restored upon readdition of external Na' or removal of amiloride. In the steady-state, addition of amiloride caused a small and slow decrease in pH, which was readily reversed upon removal of amiloride. In the presence of HC03-, removal of external C1-caused pH, to rapidly and reversibly increase by 0.23 * 0.03 (n = 15) and the initial rate of alkalinization was 20.6 * 2.7 x lo-, pWsec. In the absence of external Na+, removal of bath C1still caused pHi to increase by 0.15 * 0.02 and the initial rate of pH, increase was not significantly altered. In the nominal absence of HC0,-, removal of bath C1-caused pHi to increase very'slightly (0.05 * 0.01) with an initial dpHi/dt of only 4.4 * 0.2 x lop4 pWsec (n = 4). Addition of 100 pM DIDS did not inhibit the pH, increase caused by removal of bath C1-. These data indicate that 1) Rat Schwann cells regulate their pHi via an Na-H exchange mechanism which is moderately active at steady-state pHi. 2 ) In the presence of HC03-, there is a Na-independent C1-HC03 (base) exchanger which also contributes to regulation of intracellular pH in Schwann cells. o 1994 Wiley-Liss, Inc.