Adrenocortical secretion is up-regulated in women with polycystic ovary syndrome (PCOS), and absolute adrenal androgen (AA) excess is evident in -25% of these patients. We hypothesized that AA biosynthesis is an inherited trait and that, as for other inherited traits, AA biosynthesis remains stable over time. To test this hypothesis, we prospectively studied 23 off-treatment PCOS patients and seven age- and body mass index-matched control women on two separate occasions 3-5 yr apart (45.0 ± 19.0 months and 47.4 ± 21.3 months, respectively; P > 0.05). All subjects underwent an acute adrenal stimulation using 0.25 mg ACTH-(1-24), and dehydroepiandrosterone (DHEA), androstenedione, and cortisol (F) were measured 0 and 60 min post ACTH; basal levels of total and free testosterone (T), SHBG, and DHEA sulfate (DHEA-S) were also assessed. Among PCOS patients, the mean DHEA-S levels during the repeat study were significantly lower when compared with the initial assessment (170 ± 107 μg/dl vs. 134 ± 79 μg/dl, respectively; P = 0.02). However, only patients with initial DHEA-S levels above the median (high DHEA-S) experienced a net decrease in the levels of this metabolite (252.5 ± 99.2 μg/dl vs. 174.3 ± 82.5 μg/dl; P = 0.001) over the time of the study; patients with initial DHEA-S levels in the lower half (low DHEA-S) did not experience a change in DHEA-S (94.6 ± 28.8 μg/dl vs. 97.7 ± 56.5 μg/dl; P = 0.85). In patients, the total T levels tended to be higher at the second study, although SHBG levels were also higher, resulting in unchanged free T levels over time. Among controls, no significant changes in basal androgens were observed over the time of the study. There were no significant differences in either the basal or ACTH-stimulated levels of DHEA, androstenedione, or F over the time of the study in either PCOS or control women. We conclude that the adrenocortical secretion of AAs or F in PCOS and control women remains stable over time, supporting the hypothesis that the adrenal response to ACTH may be an inherited trait. Alternatively, a decrease in DHEA-S levels over time was observed but only among PCOS patients whose initial levels of this metabolite were above the group median, suggesting that the activity of sulfotransferase in these patients may be up-regulated by factors other than those affecting adrenocortical biosynthesis and that such regulatory influences attenuate over time.