We measured follicular fluid hormone levels in 48 normally cycling infertile women who underwent follicle puncture and oocyte retrieval during diagnostic laparoscopy at time-bracketed intervals after an endogenous LH surge. Follicular fluid LH, FSH, PRL, estrone (E1), estradiol (E2), progesterone (P), androstenedione (A), and testosterone (T) concentrations and P/E2 and A/E2 ratios were determined. Oocytes were classified as germinal vesicle (gv), metaphase I (mI), metaphase II (mII), or degenerating (dg). Follicular fluid (ff) hormone levels then were correlated with the stage of oocyte maturation. There were no differences in ff E1 or E2 levels at any stage of oocyte maturation, except that the mean ff E2 concentration was significantly (P less than 0.05) lower in ff containing dg oocytes [2,474 +/- 1,435 (+/- SE) nmol/L] than in those containing the other oocyte stages. The mean P levels were significantly (P less than 0.0001) higher in ff containing mI (48,781 +/- 10,240 nmol/L) and mII (41,801 +/- 11,098 nmol/L) oocytes than in ff containing gv oocytes (1371 +/- 696 nmol/L). The mean A level was highest (P less than 0.01) in dg-associated ff. Similarly, T was highest (P less than 0.05) in ff containing dg (52 +/- 14 nmol/L) oocytes than in ff containing mI (10.7 +/- 10.1 nmol/L) or mII (10.1 +/- 4 nmol/L) oocytes, and it was also elevated (P less than 0.05) in gv ff (72 +/- 33 nmol/L) compared to mII ff. The above differences also were reflected in the P/E2 ratio, which was significantly higher (P less than 0.05) in mI and mII ff, as well as in the A/E2 ratio, which was higher (P less than 0.05) in ff containing mI and mII oocytes compared to ff containing gv or dg oocytes. These data define the evolving changes in the microenvironment of the follicular fluid of preovulatory follicles of normally cycling women. They also provide reference points for analysis of ff obtained from women during stimulated cycles intended for in vitro fertilization.