Bone loss associated with chronic inflammatory diseases has been attributed to the release of cytokines from T lymphocytes. However, the role of T lymphocyte subsets in the mediation of osteoclast activity has not been extensively studied. Cocultures of murine bone marrow and BALC cells (murine calvarial-derived cell line) were used to study osteoclast differentiation. Murine marrow was left intact or depleted of cells expressing the CD8 or CD4 antigen by immunomagnetic separation and then cocultured with BALC cells in the presence or absence of 1,25-(OH)2D3. Depleting bone marrow of CD4-positive (CD4+) cells did not affect osteoclast differentiation (formation of tartrate-resistant acid phosphatase positive cells with three or more nuclei). However, depletion of CD8-positive (CD8+) cells resulted in a 40% increase in the number of osteoclasts formed. Addition of CD8+ cells to CD8+ cell depleted cocultures via Transwells abolished the stimulatory effects on osteoclast differentiation resulting from CD8+ cell depletion. Neutralizing antibodies to interleukin-4 and transforming growth factor-beta did not affect osteoclast differentiation in these cultures. These findings suggest that CD8+ cells may be involved in the regulation of osteoclast differentiation and that this effect is not mediated by interleukin-4 or transforming growth factor-beta.