The effect of 1,25-dihydroxycholecalciferol [1,25(OH)2D3], the active form of vitamin D3, on cell growth, clonogenicity, and cyclic adenosine monophosphate (cAMP) production was examined in human breast cancer cell line T47D. 1,25(OH)2D3 markedly inhibited proliferation of T47D cells in a time- and concentration-dependent manner. 1,25(OH)2D3 5 X 10(-7) reduced to 70% [3H]thymidine incorporation into DNA. Specific high affinity nuclear receptors for 1,25(OH)2D3 were present in this cell line. The cAMP produced by T47D cells was measured during 10 min stimulation by effectors (prostaglandin E1 or forskolin). Without effector, T47D cells produced similar amounts of cAMP in control and 1,25(OH)2D3-treated cells. After 3 days in the presence of 1,25(OH)2D3, cAMP production was significantly increased compared to control cells when stimulated by 10(-4) M prostaglandin E1 or 5 X 10(-7) M forskolin (3.2- and 2.4-fold increase, respectively). This cAMP increase was concentration dependent within the same range that inhibited cell growth and clonogenicity. These results suggest that 1,25(OH)2D3 may indirectly affect cAMP production by modulating the target cell response to stimulatory agents of cAMP production.