Previous studies have shown that C3H/HeJ (C3H) mice have higher peak bone density than C57BL/6J (B6) mice, at least in part because of differences in rates of bone resorption. The current studies were intended to examine the alternative, additional hypothesis that the greater bone density in C3H mice might also be a consequence of increased bone formation. To that end, we measured two presumptive, indirect indices of bone formation and osteoblast number in these inbred strains of mice: alkaline phosphatase (ALP) activity in serum, bones, and bone cells; and the number of ALP-positive colony-forming units (CFU) in bone marrow stromal cell cultures. We found that C3H mice had higher serum levels of ALP activity than B6 mice at 6 (118 vs. 100 U/L, p < 0.03) and 32 weeks of age (22.2 vs. 17.2 U/L, p < 0.001). Tibiae from C3H mice also contained higher levels of ALP activity than tibiae from B6 mice at 6 (417 vs. 254 mU/mg protein, p < 0.02) and 14 weeks of age (132 vs. 79 mU/mg protein, p < 0.001), as did monolayer cultures of bone-derived cells from explants of 7.5-week-old C3H calvariae and femora (8.2 times more, p < 0.02, and 4.6 times more, p < 0.001, respectively). Monolayer cell cultures prepared by collagenase digestion of calvariae from newborn and 6-week-old mice also showed similar strain-dependent differences in ALP-specific activity (p < 0.001 for each). Our studies also showed more ALP-positive CFU in bone marrow stromal cell cultures from 8-week-old C3H mice, compared with B6 mice (72.3 vs. 26.1 ALP-positive CFU/culture dish, p < 0.001). A similar result was seen for ALP-positive CFU production at 6 and 14 weeks of age, and the difference was greatest for the CFU that contained the greatest numbers of ALP-positive cells. Because skeletal ALP activity is a product of osteoblasts and has been shown to correlate with rates of bone formation, and because the number of ALP-positive CFU is believed to reflect the number of osteoprogenitor cells, the current data are consistent with the general hypothesis that bone formation may be greater in C3H than B6 mice because of a difference in osteoblast number. Our data further suggest that peak bone density may be greater in C3H mice than B6 mice due to a combination of decreased bone resorption and increased bone formation.