Spix's cavy is a potentially good experimental model for research on reproductive biology and sexual development. The aim of the present study was to evaluate the ontogeny of the steroidogenic enzymes involved in testicular androgen synthesis during prenatal development. Testes were investigated on Days 25, 30, 40 and >50 of gestation. Immunohistochemistry and immunoblotting were used to establish the site and relative amount of androgenic enzymes, including 5α-reductase, cytosolic 17β-hydroxysteroid dehydrogenase (17β-HSDI) and mitochondrial microsomal 3β-hydroxysteroid dehydrogenase (3β-HSDII), throughout prenatal development. The testicular parenchyma began to organise on Day 25 of gestation, with the development of recognisable testicular cords. The mesonephros was established after Day 25 of gestation and the ducts differentiated to form the epididymis, as testicular cords were beginning to proliferate and the interstitium to organise by Day 30 of gestation, continuing thereafter. The androgen-synthesising enzymes 5α-reductase, 17β-HSDI and 3β-HSDII were evident in Leydig cells as they differentiated at all subsequent gestational ages studied. In addition, immunoblotting showed an increase in immunoreactivity for the enzymes at Days 30 and 40 of gestation (P<0.05) and a decrease at Day 50 of gestation (P<0.05). It is concluded that the increase in androgenic enzymes in Leydig cells coincides with the functional differentiation of the testes, and with the stabilisation and differentiation of mesonephric ducts forming the epididymis.