Gallium nitrate (GN) is an inhibitor of bone resorption and thereby may result in a change in coupled bone formation. In the present investigation the effects of GN on bone formation were studied in the rat osteosarcoma (ROS) 17/2.8 cell line and normal diploid rat osteoblasts (ROB) in vitro and the femur of rats treated in vivo, measuring mRNA levels for two osteoblast parameters, type I collagen, a marker of matrix formation, and osteocalcin, a bone specific protein and also histone H4, a marker of cell proliferation. GN, at 50 μM for 3 h, increased type I collagen mRNA levels by 132% in ROS 17/2.8 cells and by 122% in proliferating ROB cells. Osteocalcin (OC) mRNA levels were decreased by 61% in ROS 17/2.8 cells and by 97% in differentiated ROB cells. These changes occurred in the absence of any effects on cell proliferation. Seventy-day-old female rats were then treated with GN, 0.5 mg/kg/day, for 3 weeks. As previously reported, GN decreased serum calcium levels, but had no effect on lumbar or femoral bone density. In contrast to the in vitro effects, GN had no effect on type I collagen steady-state mRNA levels in the femur; however, it decreased OC steady-state mRNA levels in the femur by 58%. These results suggest that GN has similar in vitro effects in transformed and normal osteoblasts, while the collagen-stimulatory effects observed in vitro cannot be extrapolated to in vivo models. The consistent inhibition of osteocalcin in vitro and in vivo suggests a more specific target for GN that may relate to its effects in inhibiting bone resorption in normal rats.