Adrenal sexual dimorphism is thought to be important in explaining sex-related differences regarding prevalent diseases and the responses to stress and drugs. We report here that in CD1 mice there is marked sexual dimorphism affecting not only gland size and corticoid hormone secretion but also adrenal ornithine decarboxylase (ODC), polyamine, and catecholamine levels in which testosterone appears to be a major determinant. Our results show that adrenal weight, ODC activity, and corticosterone and aldosterone secretion were higher in female than in male mice and that orchidectomy brought these male parameters closer to the values found in females. mRNA levels of steroidogenic proteins SF-1, Dax-1, steroid 21-hydroxylase, and aldosterone synthase appeared to be slightly higher in female than in male adrenals. Immunocytochemical analysis of adrenal ODC revealed that immunoreactivity was higher in females than in males and was located mainly in the cortical cells, and especially in zona glomerulosa, whereas no sex differences in ODC mRNA levels were observed. These results suggest that sex-associated differences in the expression of ODC in the mouse adrenal gland appear to be related mainly to posttranscriptional mechanisms. Combination treatment of mice with alpha-difluoromethylornithine (a suicide inhibitor of ODC) and a polyamine-deficient diet produced a marked decrease in adrenal polyamine and catecholamine levels and a significant reduction in plasma corticosterone and aldosterone concentrations that were not associated with a decrease in the mRNA levels of steroidogenic proteins. All of these data suggest a relevant role for testosterone, ODC, and polyamines in the mouse adrenal function.