The purpose of this study was to examine the effects of a mild 24-h stress (indwelling IV catheter) on cortisol and sleep in postmenopausal women, and to evaluate differences due to estrogen replacement therapy (ERT) status. This study, conducted in the General Clinical Research Center at the University of Washington Medical Center, examined sleep, cortisol and sleep-cortisol relationships in both baseline and stress conditions, and compared women on ERT with women not on ERT. Forty-two women (age=69.6 +/- 6.2 years [SD]), of whom 20 were on ERT, participated. Urinary free cortisol (UFC) levels and sleep polysomnography were measured over both 24-baseline and stress condition. Sleep was impaired in the stress condition for both groups; mean UFC levels were higher, sleep efficiency and minutes of stages 2, 3 and 4 sleep were reduced, and morning risetime was earlier in the stress than baseline condition. For the combined groups, age-controlled correlations between 24-h UFC and sleep were significant in both conditions: at baseline, UFC levels were associated with earlier time of rising and less REM sleep, and under stress with reduced sleep efficiency, there was reduced minutes of stages 2, 3, 4 sleep, reduced REM sleep, and an earlier risetime. The pattern of negative significant correlations between UFC and sleep/sleep timing remained when plasma estrogen was statistically controlled; however, when groups were examined separately, the significant negative UFC-sleep relationships were confined to the non ERT group. Elevated 24-h UFC is associated with impaired sleep and earlier awakening in older women not on ERT, but not in women on ERT.