In the testis, several cell types are needed to ensure male fertility, among them the testosterone-producing Leydig cells. Testosterone supports the maturation of sperm and maintains the male phenotype. Functional Leydig cells are therefore required to prevent testicular malfunction. Yet in contrast to their well-established role in the testis, the origin of the adult Leydig cell lineage has largely remained unclear. A potential reservoir for putative stem Leydig cells are the peritubular myoid cells, which show characteristics of pluripotency as well as steroidogenic capacity and can be isolated to study the differentiation mechanisms of steroidogenic cells. The scope of this work is to elucidate cells and pathways involved in the underlying processes. Common to all stages of Leydig cell development is the presence of the platelet-derived growth factor receptor alpha (PDGFRα). Stem Leydig cells have been described to be PDGFRα-positive and to not express the luteinizing hormone receptor (LHR), a classical marker for differentiated Leydig cells. To identify the stem Leydig cell in the testis, we first analyzed the two steroidogenic cell types in the postnatal rodent testis. Both, the fetal Leydig cells and the peritubular cells were PDGFRα positive and showed a transient capacity to produce steroids. Stimulation of the cAMP-PKA pathway increased the steroidogenic potential in both cell types. However, while the FLCs were responsive to hCG stimulation, the PTCs expressed no LHR and their steroid production did not increase during hCG treatment. The isolated PDGFRα-positive, LHR-negative rat PTCs expressed markers for pluripotent, steroidogenic and myoid cells, indicating that they include putative stem Leydig cells. Subsequently, we examined the transferability of the rodent data into humans. We found that the peritubular cells in the adult human testis (HTPC) also expressed the PDGFRα and showed characteristics of pluripotent as well as steroidogenic cells. Stimulation of the cAMP-PKA pathway in isolated HTPCs ...