Previous studies in microdissected rat inner medullary collecting duct (IMCD) segments have demonstrated that carbachol, arginine vasopressin (AVP), and the V₂ vasopressin receptor agonist 1-desamino-8-d-arginine vasopressin (DDAVP) induce a similar increase in intracellular Ca²⁺. The present study tested whether these agents activate the phosphoinositide hydrolysis pathway. In intracellular inositol 1,4,5-trisphosphate (IP₃) measurements, we found that IMCD suspensions incubated with AVP or DDAVP (10⁻⁸ M) displayed no measurable increase in IP₃, whereas IMCD suspensions incubated with the muscarinic cholinergic agent carbachol (100 μM) induced a significant increase in IP₃ production. Similarly, carbachol, but not AVP or DDAVP, induced a significant increase in membrane-associated protein kinase C (PKC) enzyme activity. To test what specific PKC isoforms are activated by carbachol in IMCD, we first characterized the PKC isoforms in IMCD suspensions by immunoblotting using affinity-purified antibodies against different PKC isoforms. We identified one classic PKC isoform (α), three novel PKC isoforms (δ, ε, η), and one atypical PKC isoform (ζ) in the IMCD. Carbachol induced a cytosol-to-membrane translocation of the PKC-η isoform but did not alter the distribution of any other isoform. In contrast, AVP had no effect on the distribution of any PKC isoform tested. These data, taken together, demonstrate that carbachol is an activator of the phosphoinositide hydrolysis pathway in IMCD but do not demonstrate signaling via this pathway in response to AVP or DDAVP. These results suggest that the previously observed AVP-stimulated Ca²⁺ mobilization in IMCD may be due to a mechanism other than activation of the phosphoinositide hydrolysis pathway.