Close similarity between the rat native low-conductance K⁺ channel in the apical membrane of renal cortical collecting duct principal cells and the cloned rat ROMK channel strongly suggest that the two are identical. Prominent features of ROMK regulation are a steep pH dependence and activation by protein kinase A (PKA)-dependent phosphorylation. In this study, we investigated the pH dependence of cloned renal K⁺ channel (ROMK2), wild-type (R2-WT), and PKA site mutant channels (R2-S25A, R2-S200A, and R2-S294A). Ba²⁺-sensitive outward whole cell currents (holding voltage −50 mV) were measured in two-electrode voltage-clamp experiments in Xenopus laevisoocytes expressing either R2-WT or mutant channels. Intracellular pH (pH_i) was measured with pH-sensitive microelectrodes in a different group of oocytes from the same batch on the same day. Resting pH_i of R2-WT and PKA site mutants was the same: 7.32 ± 0.02 (n = 22). The oocytes were acidified by adding 3 mM Na butyrate with external pH (pH_o) adjusted to 7.4, 6.9, 6.4, or 5.4. At pH_o 7.4, butyrate led to a rapid (τ: 163 ± 14 s, where τ means time constant, n= 4) and stable acidification of the oocytes (ΔpH_i0.13 ± 0.02 pH units, where Δ means change, n = 12). Intracellular acidification reversibly inhibited ROMK2-dependent whole cell current. The effective acidic dissociation constant (pK _a) value of R2-WT was 6.92 ± 0.03 (n = 8). Similarly, the effective pK _a value of the N-terminal PKA site mutant R2-S25A was 6.99 ± 0.02 (n = 6). The effective pK _a values of the two COOH-terminal PKA site mutant channels, however, were significantly shifted to alkaline values; i.e., 7.15 ± 0.06 (n = 5) for R2-S200A and 7.16 ± 0.03 (n = 8) for R2-S294A. The apparent ΔpH shift between the R2-WT and the R2-S294A mutant was 0.24 pH units. In excised inside-out patches, alkaline pH 8.5 activated R2-S294A channel current by 32 ± 6.7%, whereas in R2-WT channel patches alkalinzation only marginally increased current by 6.5 ± 1% (n = 5). These results suggest that channel phosphorylation may substantially influence the pH sensitivity of ROMK2 channel. Our data are consistent with the hypothesis that in the native channel PKA activation involves a shift of the pK_a value of ROMK channels to more acidic values, thus relieving a H⁺-mediated inhibition of ROMK channels.