Mechanical bending of a rat’s tibia in vivo can increase endocortical bone formation by over sixfold. It has been proposed that mechanical loading increases bone formation by driving osteoprogenitor cells in the marrow stroma to progress through the cell cycle and subsequently differentiate into osteoblasts at the cortical bone surfaces. We used a sustained-release preparation of 5-bromo-2′-deoxyuridine (SR-BrdUrd) to determine the origin of endocortical osteoblasts in rat tibiae after mechanical loading. SR-BrdUrd was bioavailable for the entire 96 h duration of the experiments, so all cells that progressed through a cell cycle were labeled with BrdUrd. Although the endocortical osteoblast surface was significantly increased (p < 0.05) at 48 h after loading, the percentage of BrdUrd-labeled osteoblasts did not increase, suggesting that the newly differentiated osteoblasts on the endocortical surface did not originate from proliferating cells. At 96 h after loading, 30–40% of the endocortical osteoblasts were BrdUrd labeled. The majority of BrdUrd-labeled osteoblasts appeared on the endocortical bone surface within the third day after loading, indicating that proliferation and differentiation of precursors into endocortical osteoblasts required 72 h after the loading stimulus. These results indicate that mechanical loading can cause two distinct osteoblastic responses:​ an immediate response within 48 h in which osteoblasts are recruited from nondividing preosteoblasts and/or bone-lining cells, and a delayed response involving proliferation and differentiation of preosteoblasts that requires ≥3 days.