We tested whether the protein kinase C (PKC) modulation of PTH-sensitive adenylate cyclase in ROS 17/2.8 cells is affected by the glucocorticoid dexamethasone and the vitamin D hormone 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. Basal and PTH- and forskolin-stimulated adenylate cyclase activities were determined in the presence or absence of 100 nM phorbol 12-myristate 13-acetate (PMA), the activator of PKC, in ROS 17/2.8 cells that had been previously cultured with or without dexamethasone or 1,25(OH)2D3. Dexamethasone treatment increased the basal, PMA-, PTH-, (PTH + PMA)- and (forskolin + PMA)-sensitive adenylate cyclase while 1,25(OH)2D3 decreased these effects. The stimulatory and inhibitory effects were dose-dependent with respect to dexamethasone and 1,25(OH)2D3, respectively. Dexamethasone increased, while 1,25(OH)2D3 decreased the maximal activity of both PTH-sensitive and PKC-modulated PTH-sensitive adenylate cyclase without affecting the half-maximal concentration (ED50) of PTH required for the activation of the enzyme. Additionally, dexamethasone, 1,25(OH)2D3 and PKC did not affect each other's ED50. Our results suggest that the effects of dexamethasone, 1,25(OH)2D3 and PKC on PTH-sensitive adenylate cyclase in ROS 17/2.8 cells are independent of each other.