Lower levels of serum bicarbonate and a higher anion gap have been associated with insulin resistance and hypertension in the general population. Whether these associations extend to other cardiovascular disease risk factors is unknown. To clarify this, we examined the association of serum bicarbonate and anion gap with cardiorespiratory fitness in 2714 adults aged 20-49 years in the 1999-2004 National Health and Nutrition Examination Survey. The mean serum bicarbonate was 24.6 mEq/l and the mean anion gap was 10.26 mEq/l, with fitness determined by submaximal exercise testing. After multivariable adjustment, gender, length of fasting, soft drink consumption, systolic blood pressure, serum phosphate, and hemoglobin were independently associated with both the serum bicarbonate and the anion gap. Low fitness was most prevalent among those in the lowest quartile of serum bicarbonate or highest quartile of anion gap. After multivariable adjustment, a 1 s.d. higher serum bicarbonate or anion gap was associated with an odds ratio for low fitness of 0.80 (95% CI 0.70-0.91) and 1.30 (95% CI 1.15-1.48), respectively. The association of bicarbonate with fitness may be mediated by differences in lean body mass. Thus, lower levels of serum bicarbonate and higher levels of anion gap are associated with lower cardiorespiratory fitness in adults aged 20-49 years in the general population.