The effects of chemically induced hypoxia and ionizing radiation on the adhesive properties of MG-63 human osteosarcoma three-dimensional spheroids were investigated. Hypoxia was induced by addition of CoCl2 to small, nonhypoxic spheroids and verified by HIF-1alpha expression. In addition, the possible role of important cell adhesion molecules involved in tumor dissemination in inducing adhesive changes were also studied. In particular, two key integrins (i.e., the alpha chain of the fibronectin receptor, alpha5, and the alpha chain of the collagen receptor, alpha2), an important member of the immunoglobulin superfamily (CD54 or ICAM-1) and the strategic molecule CD44 (H-CAM, the principal receptor for hyaluronan) were examined. Because of the important role of fibronectin in adhesive processes, variations in this extracellular matrix component were also examined. The results seem to indicate that CoCl2-induced hypoxia greatly increases adhesion of MG-63 spheroids to both tissue culture plates and plates coated with fibronectin or collagen when compared to controls, while ionizing radiation induces a great decrease in this attachment. Furthermore, chemically induced hypoxia also partially inhibits the effects of ionizing radiation. The data also show that these adhesive changes are accompanied by concomitant variations in the expression of alpha5 and alpha2 integrins, CD44, and CD54 and fibronectin.