To determine the effectiveness of retroviral-mediated purine nucleoside phosphorylase (PNP) gene transfer and expression for metabolic correction of PNP deficiency, we used as a gene transfer target the NSU-1 subline of murine S49 T lymphoma cells, an in vitro genetic model of PNP deficiency. NSU-1 cells were transduced with recombinant retroviruses that express either the murine or human PNP coding sequences under transcriptional regulation of the Moloney murine leukemia virus (Mo-MLV) long terminal repeat (LTR), resulting in expression of substantial levels of PNP activity. Untransduced or control virus-transduced NSU-1 cells were extremely sensitive to deoxyguanosine, a PNP substrate that is toxic for lymphoid cells. However, PNP-virus transduction of NSU-1 cells metabolically corrected the sensitivity of these cells to deoxyguanosine, resulting in near wild-type levels of growth inhibition. These results demonstrate that retroviral-mediated PNP gene transfer and expression corrects the metabolic defect observed in PNP-deficient murine lymphoid cells, suggesting that PNP gene transfer and expression in human lymphoid cells might similarly correct substratemediated toxicity and provide an effective genetic therapy.