Previous research identified a locus on Chromosome 14 as an important regulator of endurance exercise capacity in mice. The aim of this study was to investigate the effect of chromosome substitution on intrinsic exercise capacity and identify quantitative trait loci (QTL) associated with exercise capacity in mice. Mice from a chromosome substitution strain (CSS) derived from A/J and C57Bl/6J (B6), denoted as B6.A14, were used to assess the contribution of Chromosome 14 to intrinsic exercise capacity. All mice performed a graded exercise test to exhaustion to determine exercise capacity expressed as time (min) or work (kg m). Exercise time and work were significantly greater in B6 mice than B6.A14 and A/J mice, indicating the presence of a QTL on Chromosome 14 for exercise capacity. To localize exercise-related QTL, 155 B6.A14 x B6 F 2 mice were generated for linkage analysis. Suggestive QTL for exercise time (57 cM, 1.75 LOD) and work (57 cM, 2.08 LOD) were identified in the entire B6.A14 x B6 F 2 cohort. To identify putative sex-specific QTL, male and female F 2 cohorts were analyzed separately. In males, a significant QTL for exercise time (55 cM, 2.28 LOD) and a suggestive QTL for work (55 cM, 2.19 LOD) were identified. In the female cohort, no QTL was identified for time, but a suggestive QTL for work was located at 16 cM (1.8 LOD). These data suggest that one or more QTL on Chromosome 14 regulate exercise capacity. The putative sex-specific QTL further suggest that the genetic architecture underlying exercise capacity is different in males and females. Overall, the results of this study support the use of CSS as a model for the genetic analysis of exercise capacity. Future studies should incorporate the full panel of CSS using male and female mice to dissect the genetic basis for differences in exercise capacity.