In postmenopausal women, endogenous estradiol (E2) and free testosterone (T) have been positively associated with glucose intolerance and type 2 diabetes. Most studies have not examined these associations in a large group of postmenopausal women. The objective was to examine the association between endogenous sex hormones and glucose tolerance in postmenopausal women. This was a cross-sectional study of 1973 postmenopausal women ages 45-84 yr, not taking hormone replacement therapy, in the Multi-Ethnic Study of Atherosclerosis baseline examination. Impaired fasting glucose (IFG) and diabetes were defined based on fasting blood sugar and/or treatment for diabetes. In women with normal glucose tolerance, insulin resistance was estimated using homeostasis model assessment of insulin resistance (HOMA-IR). Increasing quartiles of bioavailable T and E2 and decreasing quartiles of SHBG were associated with significantly increased odds of IFG and diabetes (all P for trend<0.001). Except for the association of bioavailable T with diabetes, the other associations persisted after multivariable adjustment. Although higher dehydroepiandrostenedione (DHEA) was associated with greater odds of IFG (P for trend=0.02), it was not associated with diabetes. Of 1100 women with normal glucose tolerance, E2 and DHEA were positively associated, and SHBG was inversely associated with HOMA-IR (all P<0.001) after multivariable adjustment. Bioavailable T was associated with HOMA-IR (P<0.001), but not fasting glucose. Of postmenopausal women, endogenous bioavailable T, E2, and DHEA were positively associated and SHBG was negatively associated with insulin resistance.