Cardiovascular fitness represents the ability of active skeletal muscle to utilize oxygen during aerobic exercise. Elevated homocysteine, causing tissue injury by such mechanisms as oxidative stress, endothelial damage, and protein homocysteinylation, is associated with increased risk of cardiovascular disease, dementia and osteoporotic fracture. However, the association between elevated homocysteine and cardiovascular fitness has not been reported. Population-based cross-sectional study. National Health and Nutrition Examination Survey from 1999 to 2002 in the USA. A total of 1444 noninstitutionalized adults aged 20--49 years with reliable measures of cardiovascular fitness and non-missing values in homocysteine. Main outcome measures. Cardiovascular fitness, estimated maximal oxygen uptake or VO(2)max (mL kg(-1) min(-1)), was obtained by a submaximal exercise test. Levels of homocysteine were measured by the Abbott homocysteine assay, a fully automated fluorescence polarization immunoassay method and were natural-log-transformed due to right skewness. RESULTS. After adjustment for age, race and body mass index, there was a 0.70 mL kg(-1) min(-1) decrease (P=0.033) in the estimated VO(2)max for each standard deviation (SD) increase in the natural-log-transformed homocysteine level for women. Additional adjustment of hypertension, diabetes, smoking status, alcohol intake, use of lipid-lowering agents, physical activity, self-report health condition, as well as levels of folate, vitamin B(12), creatinine, C-reactive protein, total cholesterol and haemoglobin seemed to influence the association. In the fully adjusted model, we observed a 1.18 mL kg(-1) min(-1) decrease (P=0.003) in the estimated VO(2)max for each SD increase in the natural-log-transformed homocysteine level in women. There was no association between cardiovascular fitness and homocysteine levels in men. CONCLUSION. High homocysteine levels were inversely associated with cardiovascular fitness in women, but not in men. The results suggest that homocysteine levels are important indicators of exercise tolerance amongst women and may be useful in targeting female individuals requiring endurance intervention to prevent loss of cardiovascular fitness and function.