The bone formation that occurs in response to mechanical stimulation is generally considered to be a means by which bone adapts to changes in its mechanical environment. We have previously shown that the expression of genes for bone matrix proteins is maximal 72 h after a single 5‐minute episode of loading of tail vertebrae of 13‐week‐old female rats, that the predominant increase in mineralization occurs after 3 days, and that the osteogenic response to mechanical stimulation is not dependent on prior bone resorption. We have now investigated the cellular correlates of this osteogenic response. No proliferation was detected, by pulse or flash labeling, in the trabecular bone surface cells of animals killed 1 h to 10 days after the loading episode. Ultrastructural examination revealed that most of the cells covering the trabecular bone surface of control vertebrae were flat bone lining cells. After mechanical stimulation, the trabecular bone surface cells developed ultrastructural features of osteoblastic differentiation and activity, with acquisition of an increasingly cuboidal shape, rounded nuclei, and abundant rough endoplasmic reticulum. Morphometric analysis of the mean cell area, mean nuclear area, and cell and nuclear height showed that they were all maximal 48 h after loading. By 120 h after loading, the appearances of bone surface cells had reverted to those of control vertebrae. Thus, mechanical loading appears to activate lining cells, with a temporal sequence that correlates with bone matrix production.