The hormone 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] inhibited proliferation of two breast cancer cell lines (HT-39 and MCF-7) in a dose-dependent manner. We showed that 10 nM 1,25-(OH)2D3 inhibited HT-39 cell growth by 26.3 +/- 3.2% and MCF-7 proliferation by 19.5 +/- 8.6% (mean +/- SE). When both cell lines were cultured in the presence of medium containing varying concentrations of calcium, analysis of cell growth revealed that lowering the medium Ca2+ concentration from 1.3 to 0.1 mM inhibited cell proliferation of HT-39 cells by 20.0 +/- 2.3% and that of MCF-7 cells by 13.4 +/- 3.4% (mean +/- SE). Raising medium Ca2+ from 1.3 to 2.6 mM stimulated cell proliferation of HT-39 cultures by 51.8 +/- 2.3% and that of MCF-7 cells by 13.3 +/- 3.3% (mean +/- SE). When HT-39 cells were grown in medium containing different calcium levels and dosed with 10 nM 1,25-(OH)2D3, we found that the inhibitory action of 1,25-(OH)2D3 was modified. HT-39 cells in medium containing 0.1 mM calcium were more potently inhibited by 10 nM 1,25-(OH)2D3 (increased by 74% relative to that in cells in 1.3 mM calcium). Moreover, the inhibitory action of 1,25-(OH)2D3 (10 nM) on HT-39 cells grown in 2.6 mM calcium media was decreased by 68% relative to that in cells in 1.3 mM extracellular calcium. The phorbol ester 4 beta-phorbol 12,13-dibutyrate (PDB) stimulated HT-39 proliferation by 30.1 +/- 4.1%. Cells treated with PDB showed increased cell mitotic index, but unlike extracellular calcium, PDB had no effect on the dose response of 1,25-(OH)2D3 to inhibit HT-39 cell growth. We show that 1,25-(OH)2D3 action on malignant cell proliferation is dependent on the extracellular calcium concentration. The data suggest that the effect of calcium to antagonize 1,25-(OH)2D3 on HT-39 cells is not shared by all agents (e.g. PDB) that increase HT-39 cell proliferation.