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 home-based (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.