Testis development in the grey short-tailed opossum, Monodelphis domestica, was investigated by light and electron microscopy in 180 animals. On the day of birth, half the karyotyped males were found to have histologically differentiated testes. By day (d) 1 testicular cords were clearly distinguished in all XY gonads and the tunica albuginea was fully developed. At this stage the large and pale primordial germ cells could be differentiated from dark pre-Sertoli cells. From d 3 the testis became progressively rounded and testicular cords were surrounded by peritubular cells. Leydig cells were then distinguishable by the expected ultrastructural features of steroidogenically active cells, showing abundant vesicles of SER, extensive mitochondria with tubular cristae and numerous lipid inclusions. Subsequently these cells formed clusters and were surrounded by envelope cells until wk 12. Testes were located in the abdomen, attached to the large mesonephroi, until d 24 after birth when they began their descent to the scrotal sac. From 7 wk the interstitial tissue became less cellular. At the prepubertal stage (12 wk), the seminiferous tubules lacked lumina. Leydig cell cytoplasm was electron-dense with increased amounts of SER forming parallel profiles. By 4 mo (pubertal stage), seminiferous tubules were patent and various spermatogenic stages, including spermatozoa, were seen for the first time. Leydig cells then greatly outnumbered other interstitial tissue cells and were closely-packed around blood vessels but no longer clustered by envelope cells; their SER was very highly organised into masses of parallel arrays and lipid inclusions were reduced. In the adult (1 y) Leydig cells reached their greatest size; their morphological features resembled those seen at 4 mo except that lipid inclusions were sparse. In ageing Leydig cells (2-3 y), large amounts of SER were present but disorganised.