Bone is maintained by a balance between bone formation and resorption. This remodeling is controlled by a wide variety of systemic and local factors including hormones, cytokines and mechanical stresses. The present in vitro study examined the impact of medium volume, using 0.4, 0.6, 0.8, 1.0, 1.5 and 2.0 ml/well in a 24‑well plate, on the differentiation of osteoblasts and osteoclasts. There were no differences in the alkaline phosphatase activity of osteoblasts amongst the groups; however, the area of mineral deposition was decreased in a media volume‑dependent manner. A co‑culture of osteoblastic cells with bone marrow cells revealed a reduction in the total number of osteoclastic tartrate‑resistant acid phosphatase (TRAP)‑positive multinuclear cells (≥2 nuclei), whereas the formation of large osteoclastic TRAP‑positive multinuclear cells (≥8 nuclei) was increased, in a media volume‑dependent manner. There were also no differences in receptor activator of nuclear factor‑κB ligand mRNA and total osteoprotegerin (OPG) protein expression levels amongst the groups, however the concentration of OPG decreased in a media volume‑dependent manner. In conclusion, the present study demonstrated that the suppression of mineralization in osteoblastic cells and the stimulation of osteoclast fusion are dependent on the medium volume, indicating that media volume is an important factor in in vitro cell culture systems.