We report a prospective trial of in vitro sperm-mucus penetration test performed in hormonally controlled environment. To enhance the quality of the cervical mucus (CM), prevent spontaneous ovulation and to eliminate the possibility that results may be biased solely because of CM inadequacy, we conceived a 28-day physiological hormonal replacement regimen. For this women received oral estradiol valerate (E2-V) (4 mg/day) from cycle 1 to 28. CM was collected on days 13 (n = 17), 14 (n = 38) or 15 (n = 16), according to patients' individual preferences. The in vitro sperm-mucus penetration test was performed approximately 30 minutes later. On the same day, measurements of P and E2 levels were done to eliminate the possibility of spontaneous ovulation. Plasma E2 on the day of testing were 220 +/- 100 pg/ml (mean +/- SD). In all patients, except 4, P levels were low (< 1 ng/ml) on the day of the test. Our results indicate that a hormonal preparation with E2/P can be clinically useful for preventing spontaneous ovulation and reliably assuring CM quality when sperm/mucus penetration tests are performed.