Background: The effectiveness of lifestyle intervention strategies to improve blood lipids in women may be dependent on preexisting cholesterol concentrations. We characterized the effects of cholesterol status on blood lipid, lipoprotein-lipid, and lipid regulatory enzyme responses to a single session of aerobic exercise in physically active, postmenopausal women. Methods: Blood samples were obtained from twelve women with high cholesterol (HC, 200 mg/dL) and thirteen women with normal cholesterol (NC, < 200 mg/dL), 24 hr before (PRE), immediately (IPE), 24 and 48 hr after an exercise session (treadmill walking at 70 % VO_2peak, 400 kcals). Results: Repeated measures analysis revealed: 1) Preexercise cholesterol differences did not influence the lipid or lipoprotein-lipid responses to exercise; 2) for both groups, triglyceride was significantly reduced (-8.5%) after exercise, 3) the concentration profile over time for high density lipoprotein-cholesterol was significant for both groups, first falling at IPE then rising back to PRE levels by 24 hr after exercise; 4) the lecithin:cholesterol acyltransferase activity (LCATA) exercise response was group dependent increasing modestly in the NC group at 24 and 48 hr; 5) lipoprotein lipase activity (LPLA) increased at IPE (17%) in the HC group only, then fell at 24 and 48 hr (21%) compared to PRE; 6) cholesterol ester transfer protein activity was unchanged by exercise. Conclusions: In postmenopausal women, a single session of endurance exercise elicited a short-term, favorable decrease in TG independent of initial blood cholesterol concentrations. However, LCATA and LPLA postexercise changes were influenced by preexercise cholesterol status.