Studies of osteoclasts and their precursors in normal and pathological states have been severely hampered by the lack of an in vitro system for forming osteoclasts. We developed a human marrow culture system in which multinucleated cells with several characteristics of osteoclasts form. Multinucleated cells began to form during the first week of culture, with maximum numbers formed after 3 weeks. PTH (25–50 ng/ml) and 1,25-dihydroxyvitamin D3 (10^-10–10^-8M) increased formation of these cells, and these effects were inhibited by calcitonin. These multinucleated cells contained nonspecific esterase and tartrate-resistant acid phosphatase, a marker enzyme of osteoclasts, and had several ultrastructural features of osteoclasts. We used this marrow cell culture technique to study a patient with hyperparathyroidism and markedly increased osteoclasts on bone marrow biopsy. The marrow from this patient formed increased numbers of multinucleated cells in vitro. After parathyroidectomy both multinucleated cell formation in vitro and osteoclast numbers on bone biopsy decreased significantly. This long term marrow culture system represents the first demonstration of human osteoclast-like cell formation in vitro. This system should permit studies to evaluate factors controlling formation of cells with certain osteoclast characteristics in vitro and their precursors as well as to evaluate abnormalities in osteoclast formation in patients with metabolic bone disease. (Endocrinology120: 2326–2333,1987