Because cell culture systems are easily accessible for experimental genetic manipulation, male germ cell culture is of great usefulness in creating sperm vectors. This report describes that cultured male germ cells of zebrafish (Danio rerio) underwent mitosis and transmeiotic differentiation, including the entire process of meiosis, to develop into functional sperm. Enzymatically dissociated testicular cells containing germ cells were co-cultured on feeder cells derived from tumor-like testis, which exhibited features characteristic of Sertoli cells such as phagocytic activity and transcription of the Wilms' tumor suppressor wt1 and sox9a genes. Germ cells formed a clump, divided by mitosis, and differentiated into flagellated sperm on the feeders. Expression of the germ cell marker gene vas was prolonged in co-culture with the feeders, compared with culture of dissociated testicular cells alone, indicating that the feeder cells stimulate proliferation of spermatogonia. When cultured germ cells/sperm with the feeders were used for in vitro fertilization, normal embryos were obtained. Addition of the thymidine analogue 5-bromo-2'-deoxyuridine (BrdU) into culture medium resulted in BrdU-positive sperm and four-cell stage embryos after in vitro fertilization. This culture system should prove useful not only in producing transfected functional sperm, but also in analyzing the regulatory function of testicular somatic cells on the mitosis and meiosis of male germ cells in vertebrates.