The influence of arachidonic acid (AA) on the feedback regulation of mesangial contraction by large Ca²⁺-activated K⁺ channels (BK_Ca) was determined through single-channel analysis using the patch clamp method. The mesangial BK_Ca is a low-gain negative feedback inhibitor of contraction that is activated in response to agonist-induced Ca²⁺ transients and membrane depolarization. AA activated BK_Ca in cell-attached patches in a dose-dependent manner with a maximal effect at 400 nM and a half-maximal response at 49 nM. In inside-out patches, AA directly activated BK_Ca with a maximal effect at 400 nM. BK_Ca was activated significantly in response to addition of 100 nM ANG II in the presence but not the absence of AA. Since it was shown previously that fatty acids stimulated both soluble and membrane-bound guanylyl cyclase, we determined whether AA activated BK_Ca by interfering with cGMP-mediated signal transduction pathways. It was previously shown that 10 µM cGMP, via cGMP-dependent protein kinase, activated BK_Ca in a biphasic manner with an early increase in probability of a channel existing in an open state (P_o) and a subsequent inactivation mediated by protein phosphatase 2A (PP2A). We found that 10 µM dibutyryl-cGMP enhanced BK_Ca activity in an additive manner with saturating concentrations (400 nM) of AA. Moreover, the inactivation phase mediated by PP2A was not abolished. Thus AA does not affect the phosphorylation/dephosphorylation regulatory cycle for BK_Ca. It is concluded that AA potentiates the ANG II feedback response of BK_Ca by a mechanism that is independent of the phosphorylation cycle.