In single isolated rat hepatocytes Ca(2+)-mobilising hormones induce oscillations in cytosolic free Ca2+ ([Ca2+]i) in which the frequency of spiking depends on agonist dose, but the time course of individual spikes depends on the hormone species, rather than agonist concentration. We have previously presented data using sphingosine and staurosporine as evidence of a negative feedback role for protein kinase C (PKC) in the elongation of the falling phase of [Ca2+]i spikes. We show here that the principal effect of three specific PKC inhibitors, namely the bis-indolylmaleimide GF 109203X, the tetracyclic aromatic alkaloid chelerythrine, and a myristoylated PKC pseudosubstrate peptide, that act at different sites on the PKC molecule, is a reduction in, or a complete suppression of, the phenylephrine-induced [Ca2+]i oscillation frequency. These results resemble the effects of activators of PKC and modulators of diacylglycerol (DAG) metabolism. Furthermore, following phorbol ester-induced inhibition of the hepatocyte [Ca2+]i oscillator, the addition of all three of these PKC inhibitors further reduces the [Ca2+]i oscillation frequency, with high concentrations of chelerythrine being the only agent that overcomes this inhibition by phorbol ester. These paradoxical results point to the need for caution in interpreting the effects of protocols involving PKC activators and inhibitors in assessing the feedback control from PKC on cellular [Ca2+]i oscillations.