Recent evidence suggests that the biologically active testosterone includes both the free and albumin-bound fractions, while the sex hormone-binding globulin (SHBG)-bound steroid dissociates less readily. To examine the significance of the non-SHBG-bound testosterone (i.e. free plus albumin bound) in hyperandrogenism, we obtained single blood samples from 17 normal women, 20 regularly menstruating but hirsute women, and 20 oligoamenorrheic hirsute women. Each serum sample was analyzed for total testosterone by RIA, SHBG-binding capacity was determined by protein precipitation with 50% saturated (NH4)2SO4, and albumin was measured by colorimetry. Non-SHBG-bound and free testosterone and the testosterone to SHBG molar ratio were then calculated. Non-SHBG-bound testosterone was also assayed using differential protein precipitation. There were significant differences among the groups in the mean values of all variables (all P less than 0.05) except albumin. Measurement and calculation of serum non-SHBG-bound testosterone produced similar results, suggesting that the binding equation is valid. There was considerable overlap between normal (control mean +/- 2 SD) and abnormal subjects in all variables except non-SHBG-bound testosterone, for which only 3 regularly menstruating and 2 oligoamenorrheic hirsute subjects were in the normal range. As total testosterone levels increased, there was a significant increase in the ratio of non-SHBG-bound testosterone to free testosterone. These data suggest that albumin becomes increasingly more important in testosterone binding as the total serum testosterone level increases and that non-SHBG-bound testosterone may be the optimal marker to identify hyperandrogenism in hirsute women.