Epidermal growth factor (EGF), and androgen-dependent polypeptide, occurs in high concentration in male mouse submandibular gland. Glands of adult male and female mice of six inbred strains (129/J, C57BL/6J, C58/J, SWR/J, RF/J, A/J) were assayed for EGF by radioimmunoassay. In all strains, the glands of males contained 30 to 500-fold more EGF than those of females. Furthermore, significant differences in EGF content were found among the various strains in both sexes, the highest amount of EGF was present in RF/J and the lowest in C57BL/6J, with a ratio of three in the males and four in the females of the two strains, respectively. Factors that effect EGF levels were analyzed further, using these two strains. EGF was measurable in the glands of mice of both strains at 21 days of age and increased rapidly thereafter, up to 14 weeks of age. Throughout postnatal development, the level of EGF was greater in the glands of RF/J mice than in those of the C57BL/6J animals. Thirty days after castration, the EGF levels were reduced by about 98% in both strains, but the strain difference was not abolished. Testosterone implants (1 mg in Silastic tube) in castrated mice induced EGF levels six- to ten-fold compared to castrates. Even in induced animals, which had similar plasma testosterone levels, as measured by radioimmunoassays, the difference in EGF levels between the two strains was manifest. Such a difference, however, was not seen after the daily administration of 5-alpha-dihydrotestosterone for 3-14 days. Immunocytochemical straining for EGF also indicated a higher concentration of the polypeptide in the glands of RF/J mice than in those of C57BL/6J animals, and confirmed the exclusive localization of EGF in the cells of the granular convoluted tubules (GCT). According to our morphometric analysis, in the glands of male RF/J mice the GCT compartment occupied a greater portion (8% greater, P less than 0.001) of the gland volume than in C57BL/6J mice. The difference in the relative GCT volumes in the glands of female mice of the two strains was, however, statistically not significant. There was no direct correlation between the amount of EGF and the relative volume of the GCTs in the two strains. The evidence obtained implies that strain difference in submandibular-gland EGF levels are determined genetically.