Objective: The ammonia gradient from the human preovulatory follicular fluid (FF) to blood has been documented. The purpose of the present study was to substantiate whether, following controlled ovarian hyperstimulation, female sex hormones are related to this phenomenon. Material and Methods: Blood was taken from the antecubital veins of 32 randomly selected patients undergoing an in vitro fertilization program prior to oocyte retrieval and FF collection. Ammonia concentrations in blood and FF were determined by the indophenol method, and 17β-estradiol (E2) and progesterone (PGS) concentrations In plasma and FF by radioimmunoassay. Results: The mean ammonia concentration was 39.15 +/-3.25 μM for FF, and 20.15 +/-1.20 μM for blond (p <0.001). In all subjects, the ratios of ammonia concentrations in FF to those in blood were above 1.0, confirming the production of ammonia by the preovulatory follicle. No correlation was found between FF ammonia and E2 concentrations (Spearman’s rank correlation coefficient r = 0.2546; p = 0.160), nor between FF ammonia and the difference in E2 concentration in FF and plasma (r = 0.2416; p = 0.183). Similarly, there was no correlation between FF ammonia and PGS (r = -0.1089; p = 0.553). In support of this finding, no correlation was observed between FF ammonia and the difference in PGS concentration in FF and plasma (r = -0.1133; p = 0.537). Conclusions: Ammonia production is not directly related to intrafollicular female sex hormones concentrations. The accumulation of ammonia is likely to account for the alkaline pH of the human preovulatory FF.