Overexpression of manganese superoxide dismutase (MnSOD) has been postulated as one possible mechanism of radioprotection for hematopoietic cells. In this study retroviral constructs having the human MnSOD gene in both the sense and antisense orientations and the Neo-R gene as a selectable marker were transfected into the human erythroleukemic cell line K562 and the human melanoma cell line A375 by electroporation. Stably transfected K562 and A375 cells selected in G418 for 3 weeks were subjected to various doses of irradiation, and cell viability was assayed using a colony assay system in semisolid medium. Results demonstrated that K562 cells transfected with MnSOD in the antisense orientation displayed increased sensitivity to irradiation compared to parental or vector-transfected K562 cells. In contrast, A375 cells transfected with the sense MnSOD gene demonstrated increased resistance to irradiation compared to parental or vector-transfected A375 cells. The expression of the MnSOD gene in these transfected cell lines correlates with the up- or down-modulation of radiosensitivity. Thus, increased MnSOD protein was seen in the A375 cells containing the sense MnSOD, whereas decreased MnSOD protein was seen in the K562 cells containing the antisense MnSOD. These data provide evidence for the direct role of MnSOD in radioprotection using antisense gene transfer/inhibition studies.