Mice differ in their adult reproductive characteristics as a function of whether they developed in utero between two male fetuses (2M males), which have higher testosterone levels, or between two female fetuses (0M males), which have higher estradiol levels. The present study was designed to further characterize biochemical parameters of 2M and 0M adult male mice. Activities of testicular steroidogenic enzymes, namely delta 5-3 beta-hydroxysteroid dehydrogenase/isomerase, 17 alpha-hydroxylase, and C17,20-lyase (C21SCC P450), were measured by means of radiometric assays and HPLC fractionation of substrate and products. Activity of 5 alpha-reductase in both seminal vesicle and prostate was measured by similar techniques. Estrogen and androgen receptor concentrations, which indicate capacity to respond to steroid hormones, were also examined in the accessory sex organs. For both seminal vesicle and prostate, 5 alpha-reductase activities were approximately 60% greater in 2M males than in 0M males, indicating greater capacity to form dihydrotestosterone from testosterone in organs from 2M mice. No significant differences were found in testicular steroidogenic enzymes between 2M and 0M animals, whereas the trend for all three activities was higher for 2M males than for 0M males. While no differences were found in estrogen receptor concentrations, 0M prostates had three times the concentration of androgen receptors (occupied receptors) compared to 2M prostates. Our findings suggest that intrauterine fetal position exerts a significant influence on subsequent adult androgen metabolism and androgen responsiveness in reproductive organs of adult male mice.