We have reported that numerous tartrate‐resistant acid phosphatase‐positive osteoclast‐like multinucleated cells (TRAP⁺ MNCs) are formed when mouse osteoblastic cells and spleen cells are cocultured in the presence of 1α25‐dihydroxyvitamin D₃ [1α,25‐(OH)₂D₃] (Endocrinology 123:2600, 1988). In this study, we prepared a TRAP⁺ MNC population using a modified coculture system and examined its osteoclastic properties. TRAP⁺ MNCs were formed in cocultures of mouse osteoblastic cells and marrow cells on 10 cm collagen gelcoated dishes. The TRAP⁺ MNC population was prepared by treating the dishes with 0.2% bacterial collagenase followed by density gradient centrifugation. The yield of TRAP⁺ MNCs was 20,000–40,000 cells per dish, much higher than that of osteoclasts (OCLs) isolated from neonatal rat bones (∼ 1000 cells per head). The purity of TRAP⁺ MNCs was 5.6 ± 0.6% in cell number and about 30% in the number of nuclei. The recovery of TRAP⁺ MNCs after density gradient centrifugation was 30–40%. Acid production by MNCs was demonstrated by vital staining with acridine orange. Numerous resorption pits were formed when the MNC population was cultured for 48 h on bone slices. Autoradiography using [¹²⁵I]salmon calcitonin (CT) showed abundant CT binding in most TRAP⁺ MNCs. Saturation analysis of [¹²⁵I]salmon CT indicated a dissociation constant K_d for TRAP⁺ MNCs of 8.9 ± 0.7 × 10¹⁰ M and 16.5 ± 1.5 ± 10⁶ binding sites per cell. These results were similar to the value (3.5 × 10⁻¹⁰ M) and the number of binding sites (3.3 × 10⁶ per cell) in isolated rat OCLs. Displacement curves for [¹²⁵I]salmon CT with unlabeled salmon and human CT were similar in MNC and OCL preparations. Salmon and human CT increased cAMP production (maximal response: salmon CT at 10⁻¹⁰ M, human CT at 10⁻⁸ M; ED₅₀: salmon CT, 2.2 × 10⁻¹¹ M, human CT, 1.3 × 10⁻⁹ M) in the MNC preparation. These results indicate that a large number of mouse TRAP⁺ MNCs possessing OCL characteristics can be easily prepared from in vitro cultures. This procedure will facilitate examination of mammalian OCL functions.