Cortisol is a key stress hormone implicated in the pathogenesis of many age-related diseases. Longitudinal information on cortisol exposure has been restricted to animal models and a small number of human studies. The purpose of the present study was to quantify longitudinal change in cortisol across the adult life span. We conducted a prospective longitudinal study of 24-hour urinary free cortisol excretion from ages 20 to 90 years and older. Participants were 1,814 men and women from the Baltimore Longitudinal Study of Aging who provided a total of 5,527 urine specimens for analysis. The average duration of longitudinal follow-up was 6.6 years. The primary outcome measure was 24-hour urinary free cortisol to creatinine ratio (UFC/Cr) as determined by liquid chromatography/mass spectrometry. UFC/Cr follows a U-shaped pattern across the life span with decreases in UFC/Cr in the 20s and 30s, relative stability in the 40s and 50s, and increases thereafter. This pattern of change was robust with respect to adjustment for several potential confounding factors. Age-related changes in cortisol exposure raise important questions about the potential protective or exacerbating role of cortisol exposure in predicting medical, physiological, and behavioral outcomes.