Testis differentiation from representatives of the Otophysi (Cyprinus carpio), Percomorpha (Amatitlania nigrofasciata), and Atherinomorpha (Poecilia reticulata) was comparatively described. In the undifferentiated gonad of C. carpio, the primordial germ cells (PGCs) are scattered throughout the gonads while in A. nigrofasciata and P. reticulata the PGCs are restricted to the ventral periphery. In the dorsal region of the developing gonads, with the exception of C. carpio, somatic cell rearrangements result in the differentiation of the sperm duct. Pre-Sertoli cells wrap around single spermatogonia forming cysts that proliferate forming acinar-clusters. In C. carpio and A. nigrofasciata, the cysts in each acinar-cluster move away from each other, creating a central lumen. In C. carpio, the acinar-clusters then fuse to each other forming tubules that become lined by the germinal epithelium. Subsequently, the tubules anastomose dorsally and create the sperm duct. In A. nigrofasciata, the acinar-clusters elongate, forming lobules that individually connect to the sperm duct. These are lined by the germinal epithelium. In P. reticulata, the spermatogonial cysts remain in the acinar-cluster organization. Subsequently, developing ducts connect each cluster to the sperm duct and lobules subsequently develop. In the differentiated testis of C. carpio and A. nigrofasciata, spermatogonia are distributed along the lengths of the anastomosing tubules or lobules, respectively. However, in P. reticulata, the spermatogonia remain restricted to the terminal end of the lobules. Considering testis ontogeny, the spermatogonial acinar-cluster is the adult characteristic of more derived taxa that approximate the early gonad developmental stages of the basal taxa. (C) 2014 Wiley Periodicals, Inc.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Univ Estadual Campinas, Inst Biol, Grad Program Cell & Struct Biol, Campinas, SP, BrazilUNESP, Dept Morfol, Inst Biociencias Botucatu, BR-18618970 Botucatu, SP, BrazilFlorida Fish & Wildlife Res Inst, St Petersburg, FL USAUNESP, Ctr Aquicultura, Jaboticabal, SP, BrazilUNESP, Dept Morfol, Inst Biociencias Botucatu, BR-18618970 Botucatu, SP, BrazilUNESP, Ctr Aquicultura, Jaboticabal, SP, Brazil