The purpose of this study was to analyze follicular fluid (FF) samples for steroid levels from stimulated and unstimulated cycles triggered with human chorionic gonadotropin (hCG) and to assess the influence of controlled ovarian hyperstimulation and luteinizing hormone/hCG on these levels. Spontaneous ovulatory cycles were monitored with serial ultrasound examinations, and hCG 10,000 IU was given when the lead follicle was mature. Fourteen FF samples yielding fertilizable oocytes were compared with 13 FF samples from controlled ovarian hyperstimulation cycles. Progesterone (P) was higher in controlled ovarian hyperstimulation than in unstimulated cycles (9.0 +/- 1.2 micrograms/mL versus 4.4 +/- 0.6 microgram/mL; mean +/- SEM), whereas estradiol (E2) was lower (0.8 +/- 0.1 microgram/mL versus 1.3 +/- 0.2 microgram/mL), resulting in a higher P:E2 ratio (15.5 +/- 3.3 versus 4.4 +/- 0.7). Androstenedione (A), testosterone (T), and T:E2 ratios were all higher in unstimulated than controlled ovarian hyperstimulation cycles. We conclude that controlled ovarian hyperstimulation is associated with increased FF P, decreased FF E2, T, and A levels, and decreased T:E2 ratios, suggesting altered steroidogenesis and enhanced follicular aromatase activity.