Follicular fluid (FF) and oocytes were obtained from 94 follicles of 36 women for fertilization in vitro. Ovulation was induced with human menopausal gonadotropin, and follicular aspiration was performed 36 h after an ovulatory injection of hCG. The concentrations of immunoreactive hCG, FSH, and PRL were correlated with the degree of maturation of the oocyte-corona-cumulus complex mass (OCCC), fertilization, rate of cleavage, and the incidence of pregnancy after embryo transfer. Immature OCCC were derived from follicles that contained significantly lower levels of FSH than those from which intermediate and mature OCCC were derived (5.2 +/- 0.6 vs. 11.1 +/- 1.2 mlU/ml; P less than 0.05). FF from oocytes that were successfully fertilized contained higher levels of both hCG and FSH than FF surrounding oocytes that did not fertilize (136.7 +/- 8.7 vs. 108.5 +/- 10.3 mlU/ml hCG; 10.55 +/- 0.6 vs. 5.3 +/- 0.8 mIU FSH, respectively). There was no correlation between early embryonic growth rate and FF concentrations of FSH, hCG, and PRL. Ova reaching the two-cell stage 40 h after fertilization in vitro were associated with the same FF concentrations of FSH, hCG, and PRL as those that cleaved to the four-cell stage. The PRL concentration in FF was significantly higher in mature fertilized ova and in fertilized ova that were associated with a successful pregnancy. It is suggested that the intrafollicular concentration of FSH is associated with the degree of mucification of the OCCC, but FF levels of both FSH and hCG are associated with successful fertilization. High levels of PRL in FF were associated with successful pregnancy and may imply a role of this hormone in oocyte maturation.