G protein-gated inward rectifier (Kir3) channels are inhibited by activation of G(q/11)-coupled receptors and this has been postulated to involve the signaling molecules protein kinase C (PKC) and/or phosphatidylinositol 4,5-bisphosphate (PIP(2)). Their precise roles in mediating the inhibition of this family of channels remain controversial. We examine here their relative roles in causing inhibition of Kir3.1/3.2 channels stably expressed in human embryonic kidney (HEK)-293 cells after muscarinic M(3) receptor activation. In perforated patch mode, staurosporine prevented the G(q/11)-mediated, M(3) receptor, inhibition of channel activity. Recovery from M(3)-mediated inhibition was wortmannin sensitive. Whole cell currents, where the patch pipette was supplemented with PIP(2), were still irreversibly inhibited by M(3) receptor stimulation. When adenosine A(1) receptors were co-expressed, inclusion of PIP(2) rescued the A(1)-mediated response. Recordings from inside-out patches showed that catalytically active PKC applied directly to the intracellular membrane face inhibited the channels: a reversible effect modulated by okadaic acid. Generation of mutant heteromeric channel Kir3.1S185A/Kir3.2C-S178A, still left the channel susceptible to receptor, pharmacological, and direct kinase-mediated inhibition. Biochemically, labeled phosphate is incorporated into the channel. We suggest that PKC-delta mediates channel inhibition because recombinant PKC-delta inhibited channel activity, M(3)-mediated inhibition of the channel, was counteracted by overexpression of two types of dominant negative PKC-delta constructs, and, by using confocal microscopy, we have demonstrated translocation of green fluorescent protein-tagged PKC-delta to the plasma membrane on M(3) receptor stimulation. Thus Kir3.1/3.2 channels are sensitive to changes in membrane phospholipid levels but this is contingent on the activity of PKC-delta after M(3) receptor activation in HEK-293 cells.