Previous investigations established that cells isolated from the outer zone (zona fasciculata + zona glomerulosa) of the guinea pig adrenal cortex produced far more cortisol and androstenedione than those from the inner zone (zona reticularis). Studies were done to determine whether differences in microsomal metabolism might contribute to the zonal differences in steroid secretion. Cytochromes P-450 and b5 concentrations were greater in inner zone microsomes as were the magnitudes of the type I difference spectra produced by progesterone and 17 alpha-hydroxyprogesterone. Basal NADPH-cytochrome P-450 reductase activity was greater in the outer zone, but steroid substrates (progesterone, 17 alpha-hydroxyprogesterone) increased reductase activity in the inner zone and decreased activity in the outer zone. 21-Hydroxylase activity was far greater in inner than outer zone microsomes, but 17 alpha-hydroxylase activity was greater in the outer zone. As a result, progesterone was converted primarily to 17 alpha-hydroxyprogesterone by outer zone microsomes, but 11-deoxycorticosterone was the major metabolite produced by inner zone preparations. In addition, with 17 alpha-hydroxyprogesterone as substrate, the major product produced by outer zone microsomes was androstenedione, indicating relatively high C17-20-lyase activity. Inner zone microsomes by contrast, converted 17 alpha-hydroxyprogesterone primarily to the 21-hydroxylated metabolite, 11-deoxycortisol, with little production of androstenedione. The rate of conversion of pregnenolone to progesterone was also greater with outer than inner zone microsomes. The results suggest that differences in the patterns of microsomal steroid metabolism contribute to the greater secretion of cortisol and androstenedione by adrenocortical outer zone cells than by inner zone cells.