Hyposmotically induced swelling of frog renal proximal tubule cells activates a DIDS-sensitive, outwardly rectifying Cl conductance via a conventional protein kinase C (PKC). This study examines whether Na⁺-alanine cotransport similarly activates a DIDS-sensitive Cl conductance in frog renal proximal tubule cells. On stimulation of Na⁺-alanine cotransport, the DIDS-sensitive current (I_DIDS-Ala) increased markedly over time. I_DIDS-Ala exhibited outward rectification, a Na⁺/Cl selectivity ratio of 0.19 ± 0.03, and an anion selectivity sequence Br = Cl > I > gluconate. Activation of I_DIDS-Ala was dependent on ATP hydrolysis and PKC-mediated phosphorylation and was inhibited by hyperosmotic conditions. Activation could be not ascribed to a conventional PKC isoform, as I_DIDS-Ala was not affected by removing Ca²⁺ or by phorbol ester treatment, suggesting a role for a nonconventional PKC isoform, either novel or atypical. We conclude that Na⁺-alanine cotransport activates a DIDS-sensitive Cl conductance via a nonconventional PKC isoform. This contrasts with the hyposmotically activated Cl conductance, which requires conventional PKC activation.