Leydig cells (LCs) are thought to differentiate from spindle-shaped precursor cells that exhibit some aspects of differentiated function, including 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) activity. The precursor cells ultimately derive from undifferentiated stem LCs (SLCs), which are postulated to be present in testes before the onset of precursor cell differentiation. We searched for cells in the neonatal rat testis with the abilities to: (i) proliferate and expand indefinitely in vitro (self renew); (ii) differentiate (i.e., 3 beta HSD and ultimately synthesize testosterone); and (iii) when transplanted into host rat testes, colonize the interstitium and subsequently differentiate in vivo. At 1 week postpartum, spindle-shaped cells were seen in the testicular interstitium that differed from the precursor cells in that they were 3 beta HSD-negative, luteinizing hormone (LH) receptor (LHR)-negative, and platelet-derived growth factor receptor alpha (PDGFR alpha)-positive. These cells were purified from the teste; of 1-week-old rats. The cells contained proteins known to be involved in LC development, including GATA4, c-kit receptor, and leukemia inhibitory factor receptor. The putative SLCs expanded over the course of 6 months while remaining undifferentiated. When treated in media that contained thyroid hormone, insulin-like growth factor 1, and LH, 40% of the putative SLCs came to express 3 beta HSD and to synthesize testosterone. When transplanted into host rat testes from which LCs had been eliminated, the putative SLCs colonized the interstitium and subsequently expressed 3 beta HSD, demonstrating their ability to differentiate in vivo. We conclude that these cells are likely to be the sought-after SLCs.