Our aim is to examine the role of PGF_2α receptor (FP), a highly expressed prostaglandin receptor in the distal convoluted tubule (DCT) in regulating the basolateral 40-pS K channel. The single-channel studies demonstrated that PGF_2α had a biphasic effect on the 40-pS K channel in the DCT–PGF_2α stimulated at low concentrations (less than 500 nM), while at high concentrations (above 1 µM), it inhibited the 40-pS K channels. Moreover, neither 13,14-dihydro-15-keto-PGF_2α (a metabolite of PGF_2α) nor PGE₂ was able to mimic the effect of PGF_2α on the 40-pS K channel in the DCT. The inhibition of PKC had no significant effect on the 40-pS K channel; however, it abrogated the inhibitory effect of 5 µM PGF_2α on the K channel. Moreover, stimulation of PKC inhibited the 40-pS K channel in the DCT, suggesting that PKC mediates the inhibitory effect of PGF_2α on the 40-pS K channel. Conversely, the stimulatory effect of PGF_2α on the 40-pS K channel was absent in the DCT treated with DPI, a NADPH oxidase (NOX) inhibitor. Also, adding 100 µM H₂O₂ mimicked the stimulatory effect of PGF_2α and increased the 40-pS K channel activity in DCT. Moreover, the stimulatory effect of 500 nM PGF_2α and H₂O₂ was not additive, suggesting the role of superoxide-related species in mediating the stimulatory effect of PGF_2α on the 40-pS K channel. The inhibition of Src family tyrosine protein kinase (SFK) not only inhibited the 40-pS K channel in the DCT but also completely abolished the stimulatory effects of PGF_2α and H₂O₂ on the 40-pS K channel. We conclude that PGF_2α at low doses stimulates the basolateral 40-pS K channel by a NOX- and SFK-dependent mechanism, while at high concentrations, it inhibits the K channel by a PKC-dependent pathway.