We aim to examine the role of PGF2α receptor (FP), a highly expressed prostaglandin receptor in the DCT, in regulating basolateral 40 pS K channel. The single channel studies demonstrated that PGF2α had a biphasic effect on the 40 pS K channel in the DCT: PGF2α stimulated at low concentrations (less than 500 nM) while at high concentrations (above 1µM) it inhibited the 40 pS K channels. Moreover, neither 13,14-dihydro-15-keto-PGF2α (a metabolite of PGF2α) nor PGE2 was able to mimic the effect of PGF2α 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 PGF2α 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 PGF2α on the 40 pS K channel. Conversely, the stimulatory effect of PGF2α 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 PGF2α and increased the 40 pS K channel activity in DCT. Moreover, the stimulatory effect of 500 nM PGF2α and H2O2 was not additive, suggesting the role of superoxide-related species in mediating the stimulatory effect of PGF2α 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 PGF2α and H2O2 on the 40 pS K channel. We conclude that PGF2α at low doses stimulates the basolateral 40 pS K channel by NOX- and SFK-dependent mechanism, while at high concentrations it inhibits the K channel by a PKC-dependent pathway.