Cryosections of ovaries from rats treated with pregnant mare serum gonadotropin (PMSG) were immunostained with antibodies against collagen types, I, III, IV and V, laminin and heparan sulphate proteoglycan using the peroxidase-antiperoxidase technique. A uniform belt-like staining was observed between the granulosa and theca interna layers with antibodies against type IV collagen, laminin and heparan sulphate proteoglycan. Interstitial collagens type I and III stained the connective tissue in the theca externa layer but gave only faint staining in the area between the granulosa and theca interna zones. If the PMSG-treated rats were injected with human chorionic gonadotropin the number of follicles that showed a discontinuous, ragged or disrupted staining reaction in the area between the granulosa and theca interna layers was clearly increased. Surprisingly in such cases also a punctate staining reaction was seen in the basal granulosa cells. When the rats were injected with radioactive human chorionic gonadotropin, most of the ovarian follicles were labeled within 2 hours. These follicles still showed a continuous staining reaction with antibodies against basement membrane components at the border of the granulosa and theca interna cell layers. These results suggest that there is a basement membrane-like structure in Graafian follicles between the granulosa cells and theca interna layer, and that this basement membrane is disrupted after human chorionic gonadotropin stimulation.