Background & Aims: There is observational and clinical evidence that indicate that sex hormones affect development of colorectal cancer (CRC) in men and women. However, the relationship between endogenous sex hormone levels and CRC is unclear. Methods: We collected data on lifestyle, medical history, and diet etc. (through 2008), along with blood samples, from the Nurses� Health Study, the Women�s Health Study, the Health Professional Follow-Up Study, and the Physicians� Health Study II. We measured plasma levels of estrone, estradiol, testosterone, sex hormone binding globulin (SHBG), and c-peptide among 730 women (293 cases of CRC and 437 healthy individuals, as controls) and 1158 men (439 CRC cases and 719 controls), and used unconditional logistic regression to estimate relative risks (RRs) and 95% confidence intervals (CIs). All statistical tests were 2-sided. Results: Total testosterone, SHBG, and the ratio of estradiol to testosterone were associated with CRC in men after adjustments for matching and risk factors for CRC, including BMI and plasma levels of C-peptide. The RRs in the highest relative to the lowest quartile were 0.62 for testosterone (95% CI, 0.40�0.96), 0.65 for SHBG (95% CI, 0.42�0.99), and 2.63 for the ratio (95% CI, 1.58�4.36) (P-values for trend ?0.02). However, in women, only the ratio of estradiol to testosterone was (inversely) associated with CRC after adjustments for all factors (RR, 0.43; 95% CI, 0.22�0.84; P-value for trend, .03). Conclusions: Based on combined data from 4 population studies, there appears to be an association between levels of sex hormones and CRC risk in men. There also appears to be an inverse association between the ratio of estradiol to testosterone and CRC in postmenopausal women.