In the testis, Sertoli cells play a key physiological role in that they support, nourish, and protect germ cells. Because of the importance of Sertoli cells, several laboratories have established a culture system of Sertoli cells. These cultures have been well developed in mammalian species, but to our knowledge no purified avian Sertoli cells culture has been described. The aim of this study was to isolate avian Sertoli cells and to investigate their function using a chicken model in an in vitro test system. Immature chicken Sertoli cells in culture present morphology similar to that of mammalian cells and conserve expression of the specific Sertoli marker, anti-Müllerian hormone. Furthermore, in contrast to mammals, they express the 3β-hydroxysteroid dehydrogenase enzyme. Stimulation of Sertoli cells with ovine follicle-stimulating hormone rapidly activates the 3 main downstream signaling pathways of the follicle-stimulating hormone receptor: cyclic( )adenosine monophosphate/protein kinase A, phosphatidylinositol 3-kinase/Akt, and mitogen-activated protein kinase pathways. In vitro, Sertoli cells are able to secrete lactate and inhibin and have conserved the phagocytosis property. Finally, avian Sertoli cells present 3 interesting characteristics: they actively proliferate in vitro, can be passaged several times, and are suitable for freezing in nitrogen. A direct consequence of these properties is to use this cell culture test system as an alternative method to bird reprotoxicity studies.