Agents that increase intracellular cAMP (cAMP elevating agents) and 1, 25(OH)2D3 inhibit the proliferation of many cell types. We investigated the combined effect of 1,25(OH)2D3 and cAMP elevating agents on exponentially growing mouse 3T3 fibroblasts. The following cAMP elevating agents were used: theophylline and pentoxyfilline, which inhibit cAMP-dependent phosphodiesterase; prostaglandin E2 which activates adenylate cyclase by a receptor-mediated mechanism; forskolin, which directly stimulates adenylate cyclase; and the cell permeable cAMP analogs 8-bromo cAMP and N6 benzoyl cAMP. 1,25(OH)2D3 and cAMP elevating agents were added to exponentially growing fibroblasts cultured in 96-well microtiter plates and cell number was monitored 3-7 d later. 1,25(OH)2D3 and the cAMP elevating agents as single agents inhibited the growth of the 3T3 cells. The combined treatment of the fibroblasts with 1,25(OH)2D3 and the cAMP elevating agents resulted in an antiproliferative effect that was more than additive. The synergistic interaction depended on the dose of 1,25(OH)2D3 and was apparent already at 10(-8) M of the hormone. The specificity of the effect of 1,25(OH)2D3 was demonstrated by the finding that 24,25-dihydroxyvitamin D3, a vitamin D metabolite with low affinity for the vitamin D receptor, did not affect the antiproliferative effect of cAMP elevating agents. From the synergistic interaction between 1,25(OH)2D3 and the cell permeable cAMP analogs, we infer that the site of interaction between the two signaling pathways is distal to the cAMP generating and degrading machinery.