The steroidogenic enzymes 3 beta-hydroxysteroid dehydrogenase and delta 4-5 isomerase (3 beta-HSD) that convert delta 5-hydroxysteroids to delta 4-ketosteroids were measured in human follicular tissue collected during the follicular phase of the menstrual cycle. This study was done in order to determine whether 3 beta-HSD enzyme activity fluctuated during the follicular phase of the menstrual cycle and, if so, whether these changes were reflected by the concentration of steroids in the follicular fluid. A microsomal fraction was prepared from each of 14 follicular-phase follicles, and 3 beta-HSD enzyme activity was estimated by the amount of androstenedione synthesized in the presence of excess substrate (dehydroepiandrosterone) and cofactor (nicotinamide adenine dinucleotide). Androstenedione, dehydroepiandrosterone, and progesterone were measured in the aspirated follicular fluid of each follicle. 3 beta-HSD enzyme activity was undetectable in the smallest (3 to 5 mm) follicles, increased in 5 to 6 mm follicles to 363.2 +/- 60 pmol of androstenedione per milligram per hour, and increased still further in 9 to 11 mm follicles to 5,000 +/- 200 pmol of androstenedione per milligram per hour. The increase in 3 beta-HSD enzyme activity was accompanied by an increase in the concentration of androstenedione in the follicular fluid as well as of progesterone in larger follicles. These data indicate that 3 beta-HSD activity increases during the follicular phase of the menstrual cycle. It is suggested that the primary product of the increased enzyme activity is androstenedione. Since androstenedione is the principal C19 steroid produced by the ovulatory follicle and serves as a substrate for estradiol production, this increase in 3 beta-HSD activity may be important for the associated changes in the late follicular phase that lead to ovulation.