The accumulation of mast cells in the testicular interstitium of neonatally estrogen-treated rats was studied from 15 to 90 days of age. The maturation of these cells was assessed by ultrastructural analysis and their histochemical properties were examined with the sequential alcian blue-safranin staining method. The first identifiable mast cells appeared in the testis at 17-20 days of age, as immature cells with proliferative capacity. The density of mast cells increased up to 45 days of age, showing a slight decrease from 45 to 90 days of age. Before 45 days of age, most mast cells showed alcian blue-stained granules, whereas at 45 days of age, most cells presented a mixture of alcian blue and safranin-stained granules. From this age onward, most cells were stained with safranin. These maturational changes were well-correlated with their ultrastructural features. Mast cells presented few and heterogeneous immature granules up to 45 days of age, and many uniform electron-dense granules at 90 days of age. These results indicate that the testicular interstitium of neonatally estrogen-treated rats provides an advantageous environment for the recruitment, proliferation and maturation of connective tissue mast cells.