Bone formation during mechanical unloading is reduced, mainly as a result of osteoblastic hypofunction. At the same time, the total number of osteoblasts per long bone is also markedly reduced. We tested the hypothesis that the number of osteogenic precursors present in the bone marrow stroma was concomitantly diminished by using an in vitro cell culture system in which femoral adherent bone marrow cells differentiate into active osteoblasts and produce bone‐like nodules. Hindlimbs of 32‐day‐old male rats were either immobilized (unloaded) by sciatic neurectomy (immo) or sham operated (sham) and animals were killed after 11 days. Femora were either ashed to determine bone mass or used to generate bone marrow cultures. Adherent marrow cells were cultured in the presence of ascorbic acid, β‐glycerophosphate, and dexamethasone. Bone mass was significantly reduced in unloaded femora (by 16%) and tibiae (by 18%). The number of adherent cells (determined on day 6) was reduced by 50% in the immo group. Reduced cell number did not result from slower proliferation in culture since [3H]thymidine incorporation on days 4 and 6 was similar in the two groups. The osteogenic potential in vitro of marrow from unloaded bones was diminished compared with that from loaded ones as evidenced by (1) lower alkaline phosphatase (ALP) activity per mg protein (by 25–40%, examined on days 6 and 12), and (2) reduced nodule formation (by 70%, expressed as percentage of the dish area stained with Alizarin Red S on day 21). None of these changes occurred in the contralateral limb of operated (immobilized) animals. In an additional experiment, adherent cells from both groups were subcultured on day 6 and seeded at an equal density of 40,000 cells per 35 mm dish. The osteogenic potential in cultures from unloaded bones was reduced as evidenced by lower ALP activity (by 25–40%) and fewer nodules formed (by 40%). These experiments show that unloading for 11 days causes a deficit in bone mass and reduces the number of femoral adherent marrow stromal cells and their osteogenic potential in vitro in subsequent (ex vivo) cultures. They also suggest that unloading specifically reduces the number of osteogenic precursors present within the marrow stroma.