An enzymatic method of isolating primary follicles in the turkey has been described previously, but no similar work has been done in the hen. In this study, primary follicles from domestic hens (Gallus domesticus) were isolated using an enzymatic method, and the isolated follicular quantity, quality, and development in vitro were assessed. About 400 primary follicles ranging from 60 to 1125 microm in diameter were recovered with trypsin and collagenase from hen ovaries (per ovary). Of these, 76.5% were intact follicles with a complete single layer of granulose cells surrounded by the basement membrane, and their ultrastructures appeared this way in situ. Follicles (351 to 500 microm in diameter) were cultured in vitro, and 46.67% of them survived after 5 days. Ultrastructural examination showed that elongated mitochondria forming a ring were distributed to the periphery of the oocyte, the Golgi was oriented with the maturing face toward the granulosa cell layer, and the oocyte plasma membrane presented a few short microvilli lying on the oocyte surface, which confirmed that the surviving follicles were developmental. These results suggest that a simple, rapid, effective enzymatic method can be used to isolate a great number of intact primary follicles from the hen ovary.