Previous reports indicate that peak bone density is significantly higher in C3H/HeJ (C3H) than in C57BL/6J (C57BL) mice, making these two inbred strains useful models for studying the genetic basis for peak bone density. The following study was undertaken to examine whether strain differences in the bone density of C3H and C57BL mice are associated with differences in intestinal calcium (Ca) absorption. Calcium absorption was measured by the balance technique and animals received two injections of fluorochromes 5 days apart before killing. Subsequently, the femurs were removed and, following measurement of volumetric density, the left femur was divided into three equal parts and the middle third served as the femoral cortical diaphysis. Femur diaphyseal volumetric bone density, ash, and Ca content were 10%, 29%, and 29% higher in C3H than in C57BL mice (p < 0.001), respectively. Bone length, periosteal mineral apposition rate, and periosteal bone formation rate of femoral diaphyseal cortical bone were not significantly different between the two strains of mice, but the marrow area of C57BL mice was almost twofold that of C3H mice (p < 0.0001). Intestinal Ca absorption and 1,25-dihydroxyvitamin D [1,25(OH)2D]-stimulated Ca2+ uptake by intestinal mucosal cells were 38% and 51% higher in C3H than in C57BL mice p < 0.001), respectively. Serum Ca and 1,25(OH)2D levels were 6% and 32% higher in C3H than in C57BL mice (p < 0.001), respectively, and the number of intestinal-occupied vitamin D receptors was 51% higher in C3H than in C57BL mice (p < 0.01). In a second experiment, three groups of C3H mice and three groups of C57BL mice were fed diets that contained 0.4%, 0.1%, or 0.02% Ca, and serum Ca, 1,25(OH)2D, parathyroid hormone (PTH), and intestinal Ca absorption measured. At all dietary Ca levels, C3H mice maintained positive Ca absorption and absorbed significantly more Ca than C57BL mice. In contrast, at low dietary Ca levels (0.1% and 0.02% Ca), C57BL mice maintained negative Ca absorption. Low dietary Ca increased serum PTH significantly in C57BL but not in C3H mice, and decreased serum 1,25(OH)2D and Ca levels in both strains of mice. Our findings indicate that the C57BL mice relied more on the mobilization of Ca from bone to maintain extracellular Ca homeostasis than the C3H mice. We conclude that strain differences in bone mass and density between C3H and C57BL mice is expressed, in part, through the vitamin D and PTH endocrine systems and their effects on the maintenance of extracellular Ca homeostasis.