There is considerable evidence for a genetic basis underlying individual differences in spontaneous physical activity in humans and animals. Previous publications indicate that the physical activity level and pattern vary among inbred strains of mice, and identified a genomic region on chromosome 13 as quantitative trait loci (QTL) for physical activity. To confirm and further characterize the role of chromosome 13 in regulating daily physical activity level and pattern, we conducted a comprehensive phenotypic study in the chromosome 13 substitution strain (CSS-13) in which the individual chromosome 13 from the A/J strain was substituted into an otherwise complete C57BL/6J (B6) genome. The B6 and A/J parental strains exhibited pronounced differences in daily physical activity, sleep-wake structure, circadian period and body weight. Here we report that a single A/J chromosome 13 in the context of a B6 genetic background conferred a profound reduction in both total cage activity and wheel-running activity under a 14-hr light: 10-hr dark (LD) cycle as well as in constant darkness, compared to B6 controls. Additionally, CSS-13 mice differed from B6 controls in the diurnal distribution of activity and the day-to-day variability in activity onset. We further performed a linkage analysis and mapped a significant QTL on chromosome 13 regulating the daily wheel running activity level in mice. Taken together, our findings reveal a QTL on chromosome 13 with dramatic and specific effects on daily voluntary physical activity, but not on circadian period, sleep or other aspects of activity that are different between B6 and A/J strains.