We have used the patch clamp technique to study the effect of arachidonic acid (AA) on the basolateral K channels in the medullary thick ascending limb (mTAL) of the rat kidney. An inwardly-rectifying 50 pS K channel was identified in cell-attached and inside-out patches in the basolateral membrane of the mTAL. The channel open probability (P_o) was 0.51 at the spontaneous cell membrane potential and decreased to 0.25 by 30 mV hyperpolarization. Addition of 5 µM AA decreased the channel activity identified as NP_o from 0.58 to 0.08 in cell-attached patches. The effect of AA on the 50 pS K channel was specific because 10 µM 11,14,17 cis-eicosatrienoic acid had no significant effect on channel activity. To determine whether the effect of AA was mediated by AA per se or by its metabolites, we examined the effect of AA on channel activity in the presence of indomethacin, an inhibitor of cyclooxygenase (COX), or N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS), an inhibitor of cytochrome P450 monooxygenase. Inhibition of COX increased channel activity from 0.54 to 0.9. However, indomethacin did not abolish the inhibitory effect of AA on the 50 pS K channel. In contrast, inhibition of cytochrome P450 metabolism not only increased the channel activity from 0.49 to 0.83 but also completely abolished the effect of AA. Moreover, addition of DDMS can reverse the inhibitory effect of AA on channel activity. The notion that the effect of AA was mediated by cytochrome P450-dependent metabolites of AA is also supported by the observation that addition of 100 nM 20-hydroxyeicosatetraenoic acid (20-HETE), a main metabolite of AA in the mTAL, can mimic the effect of AA. We conclude that AA inhibits the 50 pS K channel in the basolateral membrane of the mTAL and that the effect of AA is mainly mediated by cytochrome P450-dependent metabolites of AA.