The aim of the present study was to determine whether the alteration of population of cells containing 3β-hydroxysteroid dehydrogenase (3β-HSD) is responsible for the formation of cystic follicles. Paraffin sections of healthy (2 to 5 mm in diameter), atretic (2 to 5 mm) and cystic follicles (more than 25 mm) were immunohistochemically stained with rabbit polyclonal antibody to bovine 3β-HSD. The 3β-HSD-positive cells were counted in 4 different regions of the follicles from the apical to the basal side. The frequencies of 3β-HSD-positive granulosa cells in cystic follicles were significantly higher than those in the healthy follicles (P<0.05), although the number of 3β-HSD-positive granulosa cells in the cystic follicle were fewer than half the cells (30 to 40%) and was much smaller than that in preovulatory follicles (Conley et al., 1995). The frequencies of 3β-HSD-positive cells were higher in the granulosa layer and lower in the theca interna layer of the cystic follicles than the atretic follicles. These results suggest that the differentiation of granulosa cells to express 3β-HSD might be insufficient in cystic follicles and accordingly they fail to ovulate. The differences of frequencies of 3β-HSD-positive cells in the granulosa and theca interna layers between cystic and atretic follicles may be one of the reasons why regression is delayed in cystic follicles.