Cells isolated from samples of human iliac crest and human femoral heads by collagenase digestion have been successfully cultured in Fitton-Jackson modified BGJ_b culture medium supplemented with penicillin (100 units/ml), streptomycin (100 Mg/ml), and fetal calf serum (10%). Although only a low proportion of the cells survived the initial plating (<1%), cells established in culture were readily passaged. Examination of cells obtained at intervals during the collagenase digestion showed that the percentage of cells that attached increased with time of digestion. Rapid sample preparation of rat bone did not substantially increase the number of cells attaching. Thus, it seems unlikely that the low survival was due to loss of viability during sample transportation and preparation. Of several media tested BGJ_b supplemented with 10% fetal calf serum supported the best growth. Population doubling time averaged 104 hr. Cultured human bone cells were assayed for alkaline phosphatase activity using the azo dye method with naphthol ASTR phosphate as the substrate. A portion of the cells (19%) demonstrated high activity in all cultures examined regardless of the passage number of the culture. Autoradiography of cells exposed to [³H]thymidine showed incorporation of the label into both alkaline phosphate-positive and -negative cells. The stimulation of cell proliferation by growth factors was studied by determining the incorporation of [³H]thymidine into DNA. The specific skeletal growth factor from human bone stimulated cell proliferation several-fold with a half-maximal effect at 5 μg/ ml. Insulin, epidermal growth factor, and a crude preparation of somatomedin C also stimulated cell proliferation.