Bone-lining cells cover the majority of trabecular bone surfaces in adult long-lived mammals. The morphology, ultrastructure, and population density of bone-lining cells was investigated in several fatty marrow trabecular bone sites in adult beagles of different ages. Although there is a low population density of bone-lining cells on bone surfaces, their total numbers greatly exceed the numbers of osteoblasts and osteoclasts found on these bone surfaces. In one of the bone sites studied, there are significantly fewer bone-lining cells in very old beagles (12–16 years old) when compared to young adult beagles (1.5–3 years old), otherwise there are no differences in the cell population that could be attributed to aging. Bone-lining cells are flattened against bone surfaces and have flat or sometimes ovoid-shaped nuclei which are often located adjacent to areas in the fatty marrow where capillaries are found. When viewed in the electron microscope, bone-lining cells contain few organelles, and the attenuated cytoplasm of these cells is well extended over bone surfaces. The bone-lining cell cytoplasm does not appear to form a continuous layer over the bone surface, as numerous gaps and spaces are seen. Bone-lining cell processes are frequently joined by junctions morphologically similar to gap junctions. Between the lamina limitans of the bone matrix and the bone-lining cell, as well as between the bone-lining cell and the adjacent fat cells, there are layers of connective tissue containing collagenous fibers and other amorphous material. These findings are discussed in relation to the possible role of bone-lining cells in the regulation of mineral homeostasis.