The sources of cholesterol for testosterone production were investigated in freshly isolated murine Leydig cells. In vitro stimulation with human CG (hCG) (0.2 IU/ml) caused a 75-fold increase in testosterone production. Leydig cells contained approximately equal amounts of free and esterified cholesterol (7.8 vs. 8.7 micrograms/mg protein). The total cholesterol content of cells stimulated for 4 h with hCG was significantly decreased compared with unstimulated cells (8.4 vs. 17.6 micrograms/mg protein); both free and esterified cholesterol decreased by about 50%. In unstimulated Leydig cells incubated with [14C]acetate for 12 h, the majority of incorporated [14C] was found in free and esterified cholesterol, whereas, in the hCG-stimulated cells, 80% of incorporated 14C was in testosterone. The activity of hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase after 4 h in hCG-stimulated cells was 20% higher than in unstimulated cells (115.5 vs. 84.4 pmol/mg protein.min). However, by 6 h, HMG-CoA reductase activity doubled in the hCG-stimulated compared with unstimulated cells. By 12 h, HMG-CoA reductase activity in hCG-stimulated cells was 4 times the preincubation level and 8 times the 12-h level in unstimulated cells. HMG-CoA reductase activity induced by hCG was blocked by aminoglutethimide, an inhibitor of the cholesterol side-chain cleavage enzyme. Lovastatin, a potent inhibitor of HMG-CoA reductase, had no effect on unstimulated or hCG-stimulated testosterone production during a 12-h incubation. Murine high density lipoproteins (mHDL) increased HMG-CoA reductase activity in both unstimulated (29%) and hCG-stimulated (20%) cells. During a 6 h incubation, mHDL increased hCG-stimulated testosterone production by 20%, but had no effect on unstimulated testosterone production. These results suggest that murine Leydig cells store enough cholesterol and cholesteryl esters to support testosterone production for at least 12 h in vitro. Although mHDL does not have a major stimulatory effect on testosterone biosynthesis, it may be involved in the regulation of de novo cholesterol synthesis.