Heterotopic ossification is a common complication in which bone develops in soft tissues. Although frequently benign, in some patients the condition becomes painful, restricts motion, and requires surgical intervention. This condition and the cells responsible for it are poorly characterized. Using cell culture techniques, this study compares the performance of osteogenic cells obtained from heterotopic ossification with trabecular osteoblasts isolated from the same patient. Cells isolated from trabecular bone samples and heterotopic ossification sites from six patients were evaluated for osteocalcin production after exposure to 1,25-dihydroxycholecalciferol, alkaline phosphatase activity, typing and synthesis of collagen, cell proliferation, and total protein content. Samples of heterotopic ossification and trabecular bone from three of the patients were examined histologically. Heterotopic ossification derived cells were shown to produce osteocalcin, Type 1 collagen, and alkaline phosphatase activity. They also had increased rates of collagen synthesis, alkaline phosphatase activity, and cell proliferation compared with the normal osteoblasts. Initial tissue from the heterotopic ossification sites showed increased numbers of osteocytes/mm2 compared with normal trabecular bone. Although heterotopic ossification derived cells functioned qualitatively like osteoblasts, they exhibited elevated levels of activities traditionally ascribed to osteoblasts, such as collagen synthesis and alkaline phosphatase activity.