The preovulatory human follicular fluid contains only HDLs as a lipoprotein class with a typically high proportion of prebeta HDL. We first examined the role of follicular fluid and HDL subfractions on human spermatozoa capacitation, a process characterized by a hyperactivation of the flagellar movement and a depletion of plasma membrane cholesterol. Whole follicular fluid and isolated HDL, used at constant free cholesterol concentration, were both able to promote an early flagellar hyperactivation. Moreover, incubation of [(3)H]cholesterol-labeled spermatozoa with follicular fluid induced a rapid cholesterol efflux from spermatozoa that was confirmed by mass measurements of cholesterol transfer. Using isolated HDL, the cholesterol efflux had a similar time course and represented 70% of that mediated by whole follicular fluid. We then analyzed the time course of radioactive labeling of HDL subfractions. In the first minute of incubation, we found that the prebeta HDL fraction incorporated the main part of the radioactivity (60%), with the rest being found in alpha-HDL, but strikingly, the labeling of alpha-HDL increased with time at the expense of prebeta HDL.Thus, our results indicate that HDLs are involved in both spermatozoa hyperactivation and cholesterol effl ux and suggest the role of prebeta-HDL particles as fi rst cellular cholesterol acceptors.