Prolactin is associated with an increased risk of postmenopausal breast cancer; however, few modifiable factors are known to reduce prolactin concentrations. Therefore, we examined the effect of a 12-month moderate-intensity exercise intervention on serum prolactin concentrations as a secondary end point (primary end points were estrogens and androgens). We randomly assigned 173 postmenopausal women who were sedentary, overweight (body mass index >24 kg/m 2 , body fat >33%), ages 50 to 75 years, and not using hormone therapy to an exercise intervention or stretching control group. The intervention was facility-and homebased (45 min, 5 days/wk moderate-intensity sports/recreational exercise). One hundred and seventy (98%) women completed the study. Prolactin concentrations were similar at baseline (P = 0.25, geometric mean exercisers = 6.9 and controls = 7.5 ng/mL). Overall, the intervention was not associated with changes in prolactin concentrations between exercisers and controls at 3 months (P = 0.46) or 12 months (P = 0.29). The intervention effect did not vary by baseline age, body mass index, parity, or change in percent body fat during the intervention. Among exercisers, there was a significant difference in prolactin concentrations by change in fitness (VO 2 max) between baseline and 12 months. Exercisers whose VO 2 max changed by <5% had a 5% increase in prolactin concentrations, whereas those who increased their VO 2 max by 5% to 15% and >15% had a 11% (P = 0.03) and 7% (P = 0.01) decrease in prolactin concentrations, respectively. Although the exercise intervention had little effect on prolactin concentrations overall, increasing physical fitness was associated with reduced prolactin concentrations among postmenopausal women. (Cancer Epidemiol Biomarkers Prev 2007;16(5):895 -9)