A previous study conducted in rat-1 cells expressing alpha(1A)-adrenergic receptors showed that phenylephrine (PHE) stimulates phospholipase D (PLD) activity. This study was conducted to determine the contribution of protein kinase C (PKC) to PHE-induced PLD activation in these cells. PKC inhibitors bisindolylmaleimide (BIM) I and Ro 31-8220, but not Gö 6976 or a pseudosubstrate peptide inhibitor of PKCalpha, decreased PLD activity and arachidonic acid release elicited by PHE. However, antisense oligonucleotides directed against PKC alpha, delta, epsilon, and eta reduced PKC isoform levels by about 80% but failed to alter PHE-induced PLD activation, indicating that these PKC isoforms are not involved in PLD activation elicited by alpha1A-adrenergic receptor stimulation. Ectopic expression of a kinase-deficient mutant of the PKC-related kinase PKN significantly attenuated PHE-induced PLD activation. On the other hand, BIM I and Ro 31-8220 blocked PHE-mediated increase in intracellular Ca2+ but Gö 6976 and the peptide inhibitor did not. In the absence of extracellular Ca2+, PHE failed to increase PLD activity. These results indicate that alpha1A-adrenergic receptor-stimulated PLD activation is mediated by a mechanism independent of PKCalpha, delta, epsilon, and eta, but dependent on a PKC-related kinase, PKN. Moreover, PKC inhibitors BIM I and Ro 31-8220 block PHE-induced PLD activity by inhibiting calcium signal. Caution should be used in interpreting the data obtained with PKC inhibitors in vivo.