Prolonged treatment with oxolinic acid is known to elevate serum luteinizing hormone (LH) levels, resulting in induction of Leydig cell tumors in rats. In a carcinogenicity study of the compound, tubular atrophy of the testis was also increased, suggesting that oxolinic acid might affect spermatogenesis. The present study was therefore performed using rats of different ages with a particular focus on seminiferous tubule alteration and its relation to Leydig cell proliferation. Young adult (7 weeks of age) and aged (52 weeks of age) males of the Wistar strain were administered oxolinic acid at dietary concentrations of 0 (basal diet), 300, 1000 or 3000 ppm for 4 (all groups), 13 (0 and 3000 ppm groups), 26 (0 and 3000 ppm groups), or 52 weeks (0 and 3000 ppm groups of aged rats). Serum LH levels were elevated in both young adult and aged animals treated with 3000 ppm at most examined time points. While testosterone levels were also increased at the early time points in young adult, this was not the case in older animals. Elevation of the incidences of foci and/or focal hyperplasia of Leydig cells was noted but was only slight limited to aged rats treated with 3000 ppm after 26 weeks. Furthermore, it did not appear to be related to seminiferous tubular alteration. No treatment-related histopathological abnormalities could be detected in any treatment group, and morphometrical stage analysis of spermatogenesis conducted for the control and 3000 ppm-treated groups demonstrated no lesions. These results provide strong evidence that prolonged oxolinic treatment does not directly induce testicular toxicity or altered spermatogenesis in either young adult or aged rats, except for slight increase of Leydig cell proliferative lesions caused by elevated serum LH levels. Aged rats might have higher sensitivity than young adults to the effects of oxolinic acid on proliferative lesions of Leydig cells.