In this study, we demonstrate the effect of follicular fluid (FF) on free cytosolic calcium ion ([Ca2+]i) in human spermatozoa, using a fluorescent indicator "fura 2," and we evaluate the factors that increase [Ca2+]i in FF. A significant and immediate [Ca2+]i increase (656.3 +/- 148.6 nM) was elicited by intact FF. The increase in [Ca2+]i elicited by lipid-stripped FF was only 50% (322 +/- 68.7 nM) of that elicited by intact FF. However, 82% of the [Ca2+]i increase elicited by intact FF was observed when lipid-stripped FF was supplemented with progesterone, but not when it was supplemented with estradiol. Also, progesterone at the concentrations of 1 ng/ml to 1 microgram/ml caused a significant increase in [Ca2+]i by itself, but estradiol produced a small effect, ranging from 3% to 9% of the effect produced by progesterone. These results indicate that progesterone plays a major role in the lipid fraction in FF to elicit the entry of calcium into human spermatozoa.