Phosphatidylinositol 3-kinase (Pl3-k) is involved in cellular signaling via the phosphoinositol pathway leading to mitogenesis in response to growth factors in proliferating cells, as well as cytoskeletal changes and secretory responses in terminally differentiated cells. The fungal metabolite, wortmannin, is a potent and selective inhibitor of Pl3-k at nanomolar concentrations. We show that wortmannin dose-dependently (0.001-1 microM) inhibits bone resorption by isolated rat osteoclasts in the bone slice pit assay with an IC50 of approximately 5 nM. Wortmannin was not cytotoxic since osteoclast morphology and survival on bone slices was unaffected by concentrations up to 1 microM. Since primary osteoclasts are terminally differentiated cells and osteoclast cytoplasmic spreading and morphology was unaffected by wortmannin, we suggest that Pl3-k signaling is involved in vesicle exocytosis and ruffled border membrane formation that are required for osteoclastic bone resorption to take place.