Evidence was sought for the presence of osteoclasts or preosteoclasts in cells prepared from neonatal murine calvaria by sequential enzymatic digestion. Freshly isolated cells released late in the digestion process resorbed both living and devitalized calvarial bone matrix in response to PTH, accompanied by the development of osteoclasts. Light and scanning electron microscopy of these cells after 1 to 2 days in culture revealed the presence of round cells (10 to 15 mu in diameter) with minimal surface microvilli in addition to the larger osteoblastic cells. Few cells contained tartrate-resistant acid phosphatase (TRAPase). If initially seeded at confluent density, more cells positive for TRAPase developed during subsequent culture, and these cells retained the ability to resorb calvarial bone matrix upon treatment with parathyroid hormone (PTH). These cultured cells responded to PTH with increased secretion of TRAPase into the medium and increased numbers of TRAPase positive cells. These were 20 to 50 mu in diameter, sometimes multinucleated, and some were spread 100 to 200 mu in length. Observations of living cells that took up neutral red showed that, upon treatment with calcitonin (CT), surface filopodia of some but not all of the labeled cells retracted within 30 minutes. Loss of resorptive response to PTH, as well as PTH-stimulated development of TRAPase-positive cells, occurred if the cells were initially seeded at low density and grown to confluence before exposure to hormone. This correlated with the loss of most of the 10 to 15 mu diameter round cells. These observations suggest that preosteoclasts are present among these small cells that can give rise to osteoclasts upon treatment with PTH.