We have studied the effect of exercise on the percentage of free plasma estradiol 17-beta (E2) and testosterone (T) in 13 untrained (UT) and 8 trained (marathon runners, MR) eumenorrheic women. All women performed a standardized bicycle ergometer (UT) or treadmill (MR) test in the follicular and luteal phases of their menstrual cycles (15-min consecutive work loads of 60%, 70%, and 80% VO2 max to exhaustion). Blood was drawn through an indwelling venous catheter at 15 and 2 min before and immediately after exercise. Free E2 and T was assayed by centrifugal ultrafiltration using undiluted plasma at 37 degrees C (basal samples) and 39 degrees C (exercise samples). Statistical analysis was performed by a two-tailed paired t test. In the UT, the basal percentage of free E2 was measured to be 1.55% +/- 0.24% (mean +/- SD) in the follicular phase and 1.62% +/- 0.32% in the luteal phase (differences NS). In the MR, these values were 1.22% +/- 0.25% and 1.25% +/- 0.12% for the follicular and luteal phases, respectively. The differences between the groups were not significant. Exercise did not provoke significant changes in free E2 in either of the groups. Constrastingly, exercise induced a significant (P less than 0.02) increase in the percentage of free T from 1.56% +/- 0.27% to 2.1% +/- 0.36% and from 1.6% +/- 0.27% to 2.12% +/- 0.33% in the UT in the follicular and luteal phases, respectively. In the MR, the free percentage of T increased from 1.04% +/- 0.39% to 1.6% +/-0.16% (follicular phase) and from 1.24% +/- 0.22% to 1.67% +/- 0.18% (P less than 0.02).