The association of sex hormone levels with mortality over a median of 16 years of follow-up was evaluated in a prospective cohort study. The study included 1,114 US men who participated in phase 1 (1988-1991) of the Third National Health and Nutrition Examination Survey Mortality Study and had no history of cardiovascular disease or cancer at baseline. Multivariable adjusted hazard ratios for all-cause mortality associated with a decrease in hormone concentration equal to the difference between the 90th and 10th percentiles of the sex hormone distributions were estimated by using proportional hazards regression. The hazard ratios associated with low free testosterone and low bioavailable testosterone levels were 1.43 (95% confidence interval (CI): 1.09, 1.87) and 1.52 (95% CI: 1.15, 2.02), respectively, for follow-up between baseline and year 9; they were 0.94 (95% CI: 0.51, 1.72) and 0.98 (95% CI: 0.56, 1.72), respectively, for follow-up between year 9 and year 18. Men with low free and bioavailable testosterone levels may have a higher risk of mortality within 9 years of hormone measurement. Future studies should be conducted to fully characterize the association of low free and bioavailable testosterone concentrations and mortality in men and to describe the mechanism underlying the association.