cAMP, estradiol (E2), and progesterone levels were determined in 24 follicular fluid samples obtained from 8 women who conceived after in vitro fertilization and in 47 samples from 26 women who did not. Follicular development was induced by human menopausal gonadotropin, and maturation of retrieved oocytes was assessed by the degree of cumulus mucification and corona dispersal. The mean follicular fluid cAMP concentration was significantly (P less than 0.001) lower in women who became pregnant than in those who did not (106 vs. 241 pmol/ml), while the mean E2 level was significantly (P less than 0.01) higher (727 vs. 497 ng/ml), and the progesterone to E2 ratio was significantly (P less than 0.05) lower (9.5 vs. 18.0). Overall, follicles of immature, intermediate, and mature oocytes did not differ in cAMP content. However, intermediate and mature oocytes from women who became pregnant were derived from follicles containing significantly (P less than 0.01) lower cAMP levels than those of women who did not become pregnant (66 and 122 vs. 233 and 288 pmol/ml, respectively). Furthermore, fertilized oocytes leading to conception originated from follicles with significantly (P less than 0.001) lower cAMP concentrations than the follicles that yielded nonfertilized oocytes or fertilized oocytes not leading to conception (92 vs. 270 and 240 pmol/ml, respectively). Similarly, significantly (P less than 0.05) lower cAMP levels were found in the follicular fluid of cleaved oocytes resulting in a pregnancy compared to those that did not (86 vs. 236 pmol/ml). It is concluded that low levels of cAMP are associated with successful fertilization and cleavage of human oocytes in vitro resulting in viable pregnancies and may, therefore, be used as a marker of optimal follicular development in in vitro fertilization cycles.