Recently, the protein kinase C (PKC) activator 12-O-tetradecanoylphorbol 13-acetate (TPA) was found to have prolactin (PRL)-like actions on specific metabolic processes in mouse mammary gland explants. Since TPA is known to stimulate PKC, these observations suggest that PKC may have a role in the PRL stimulation of lactogenic processes. The present studies provide further evidence for this by demonstrating a transient, time-dependent translocation of PKC to the particulate fraction in response to PRL. Particulate-associated PKC reached a maximum between 15-30 min and returned to control values within 1-2 h after PRL treatment. PRL treatment for 16 h also induced a down-regulation of total cellular PKC. Inhibition of PKC function, either by a 30 h pretreatment with TPA (PKC down-regulation) or 2 h with 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7), resulted in an attenuation of PRL-stimulated effects on ornithine decarboxylase activity and synthesis of RNA, caseins, and lipids.