The effects of phorbol 12-myristate 13-acetate (PMA), a known activator of protein kinase C, on receptor-mediated stimulation of adenylate cyclase were evaluated in a rat osteosarcoma cell line (UMR-106) with the osteoblast phenotype. Pretreatment of UMR-106 cells with PMA increased parathyroid hormone (PTH)-stimulated adenylate cyclase activity and inhibited prostaglandin E2 (PGE2)-responsive enzyme activity. In addition, PMA enhanced enzyme activation by forskolin, which is thought to exert a direct stimulatory action on the catalytic subunit of adenylate cyclase. The regulatory effects of PMA were concentration dependent and of rapid onset (less than or equal to 1 min). Treatment with PMA also resulted in translocation of protein kinase C activity from the cytosol to the particulate cell fraction. Pertussis toxin, which attenuates inhibition of adenylate cyclase mediated by the inhibitory guanine nucleotide-binding regulatory protein (Gi), augmented PTH-sensitive adenylate cyclase activity and reduced the incremental increase in PTH response produced by PMA. The results suggest that activation of protein kinase C increases PTH-stimulated adenylate cyclase activity by actions on Gi and/or the catalytic subunit and decreases PGE2 responsiveness by a mechanism involving the PGE2 receptor.