Results of in vitro experiments indicate that with increasing concentrations of SHBG, testosterone (T) is preferentially bound to SHBG in comparison with estradiol (E2). In these studies, the ratio of non-SHBG-bound E2 (non-SHBG-E2) to non-SHBG-T increased with increasing levels of SHBG. SHBG has consequently been regarded as an estrogen amplifier. In this cross-sectional study in 399 men aged between 40 and 80 yr we tested whether higher levels of SHBG are associated with a higher estrogen/androgen ratio in vivo. The mean T level of these men was in the eugonadal range [536 ± 152 ng/dl (18.6 ± 5.26 nmol/liter), mean ± SD]. With increasing SHBG levels the non-SHBG-bound fraction of T decreased from 80 to 36% and that of E2 from 89 to 53%. Higher levels of SHBG were associated with higher levels of both total T [regression coefficient (β) after adjustment for age and body mass index, 286 ± 15.8; P < 0.001] and total E2 (β = 4.47 ± 0.90; P < 0.001). However, SHBG levels were negatively related with levels of non-SHBG-E2 (β = -1.78 ± 0.69; P < 0.001), whereas there was a positive association between levels of SHBG and non-SHBG-T (β = 32.0 ± 9.78; P = 0.001). Furthermore, we observed a negative relationship between SHBG levels and the E2/T ratio of either total (β = -0.016 ± 0.002; P < 0.001) or non-SHBG-bound (β = -0.011 ± 0.002; P < 0.001) hormone. Therefore, we conclude that in eugonadal men, higher SHBG levels are associated with lower levels of non-SHBG-E2 but slightly higher levels of non-SHBG-T. This means that SHBG cannot be regarded as an estrogen amplifier in eugonadal men.