The patch-clamp technique was used to identify individual Na channels in the apical membrane of the rat cortical collecting tubule and to evaluate the effects of cytoplasmic Ca2+ and pH on channel activity. In excised, inside-out patches, the probability of a channels's being open (P0) increased with alkalinization of the solution bathing the cytoplasmic side of the patch. Estimates of P0 were 0.05 at pH 6.4, 0.19 at pH 6.9, and 0.41 at pH 7.4. Varying the free Ca2+ concentration of the solution bathing the cytoplasmic side of the patch had no measurable effect on P0. In cell-attached patches, addition of the Ca2+ ionophore ionomycin to the solution bathing the tubules to a final concentration of either 1 or 10 microM decreased channel activity measured as the mean number of open channels (no. open) = n X P0 where n is the number of channels in the membrane. (no. open) was significantly decreased at 3 min after addition of ionomycin and fell to less than 10% of control values after 10 min incubation. There was no fall in (no. open) either in time controls or in tubules exposed to ionomycin in the presence of low bath Ca2+ concentrations [no added Ca2+ with 1 mM ethyleneglycol-bis-(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA)]. The results suggest that cytoplasmic pH can directly influence channel activity. Cytoplasmic Ca2+ does not interact directly with the channels, but increased cytoplasmic Ca2+ produces a fall in channel activity through an indirect process.