A rapid and simple technique for assessing ovulation and luteal function is needed in clinical practice. A sensitive immunoassay kit is presented here for clinical use. An immunoassay of pregnanediol-3-glucuronide (Pd-3G) in urine was established. The sensitivity of this assay was 0.4 mg/l of Pd-3G. The standard error range of the recovery test was within 0.8 mg/l, and the coefficients of variation of within-run and day-to-day precision test were within 4%. Cross reactions of various steroids were observed in free pregnanediol, pregnanetriol and progesterone, but their reactivity exhibited less than 4%. The Pd-3G/creatinine (mg/g) ratio was demonstrated as nearly constant over a 24-hour period. A significant rise of Pd-3G coincided with a luteinizing hormone surge and continued during the luteal phase in the ovulatory menstrual cycle. In an anovulatory cycle, Pd-3G was found to be under 4 mg/g creatinine. This Pd-3G direct-assay method could be widely used in infertility clinics.