Treatment of logarithmically growing rat intestinal epithelial cells (IEC-6) in culture with vitamin D3 (cholecalciferol), 25-hydroxy vitamin D3 (25-hydroxy cholecalciferol), 1,25-dihydroxy vitamin D3 (1,25-dihydroxycholecalciferol), and 24,25 dihydroxy vitamin D3 (24(R),25-dihydroxycholecalciferol), caused an inhibition of the cholesterol biosynthetic pathway at two separate sites. At concentrations greater than 2 micrograms/ml, the hydroxylated forms of vitamin D3 caused an accumulation of methyl sterols indicating an inhibition of lanosterol demethylation. Vitamin D3, however, had little effect on lanosterol demethylation. A second site of inhibition occurs at 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase), the rate limiting enzyme in cholesterol biosynthesis at concentrations less than 2 micrograms/ml. All vitamin D3 compounds, except 1,25-dihydroxy vitamin D3, inhibited HMG-CoA reductase activity in a concentration-dependent manner. The lack of inhibition of HMG-CoA reductase activity by 1,25-dihydroxy vitamin D3 in IEC-6 cells was not due to impaired uptake, since 1,25-dihydroxy vitamin D3 caused an accumulation of methyl sterols under similar conditions. The inhibition of HMG-CoA reductase activity and cholesterol synthesis by vitamin D3 and 25-hydroxy vitamin D3 was also observed in other cell culture lines such as human skin fibroblasts (GM-43), transformed human liver cells (Hep G2), and mouse peritoneal macrophages (J-774). On the other hand, 1,25-hydroxy vitamin D3 showed effects on HMG-CoA reductase activity that varied with the cell line. In J-774 and human skin fibroblasts, 1,25-dihydroxy vitamin D3 showed a biphasic effect on reductase activity such that at low concentrations reductase activity was inhibited but was restored to control values at high concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)