There are three possible sources of cholesterol for immediate use in progesterone production by preovulatory human granulosa cells: follicular fluid high-density lipoprotein, de novo synthesis of cholesterol, and performed intracellular cholesteryl ester stores. In the present study these three alternatives were investigated. First, an in vitro model was established that mimics the preovulatory environment, including short-term cultures and use of autologous follicular fluid in the culture medium, instead of serum. Using this model it was found that the presence of high-density lipoprotein from follicular fluid in the culture medium did not affect the synthesis of progesterone by the granulosa cells. Next, addition of inhibitors of de novo sterol synthesis, like low-density lipoprotein, 25-OH cholesterol and compactin to the culture medium, did not reduce [14C]acetate incorporation into sterols and steroids by the cells. The sterol synthesis was accordingly interpreted to be at a low and therefore uninhibitable level. Finally, the content of free and esterified cholesterol in freshly isolated granulosa cells was found to be 50 +/- 7 and 52 +/- 13 pmol/mg cell protein, respectively. We suggest that neither follicular high-density lipoprotein nor endogenous synthesis is the immediate cholesterol source for the progesterone production in preovulatory human granulosa cells. However, granulosa cells have a large store of cholesteryl esters that may provide free cholesterol for the preovulatory progesterone production.