A method is presented for separating osteoclasts from the heterogeneous population of bone and marrow cells. Cell suspensions were prepared from femora of young rabbits by mechanical dispersion. The starting cell suspension typically contained only 1.0% +/- 0.5 osteoclasts. Following an initial 45 min of unit gravity sedimentation in a lucite chamber osteoclasts were primarily distributed in fractions 2-5. A second 45-min sedimentation of these pooled fractions yielded cell suspensions containing greater than 30% osteoclasts (as much as a 50-fold increase over starting percentages). Linear scan analysis, however, revealed that osteoclasts accounted for 73.14% +/- 0.58 of the cell colume. Subsequent in vitro experiments demonstrated linear incorporation of 3H-leucine into TCA precipitable protein for cells comprising the osteoclast fraction. Concomitant radioautographs revealed radioactive label in isolated osteoclasts.