Mouse fibroblasts do not ordinarily express components for the interleukin-2 receptor (IL-2Rα, β, and γ). An analysis of these cells by reverse transcription followed by polymerase chain reaction, however, indicates the presence of transcripts specific for the IL-2R β and γ genes. Transfection of the cDNA for the α chain of the human IL-2R into LTK− mouse fibroblast cell line (L3 cells) leads, in long-term cultures, to the formation of transcripts of endogenous mouse IL-2, IL-2Rα and β genes, as detected by Northern blotting. Based upon the results of the binding of 125I-labeled IL-2 to the transfected cells, three IL-2-binding proteins of 55 kDa, 65 kDa and 75 kDa were expressed by the transfected cells. The 65-kDa and 75 kDa proteins bound IL-2 in the presence of monoclonal antibodies for the IL-2Rα chain. These polypeptides assembled to form high-affinity IL-2R, as shown by Scatchard binding analyses. The receptors were functionally active, since the expression of H-2k major histocompatibility complex antigens on the surface membranes of L3 cells was enhanced by exposing the cells to IL-2. Activation of the IL-2 gene was also observed in long-term cultures of Lαβ cells, another LTK− transfectant expressing the human IL-2Rα chain. This type of gene activation was not observed in LTK− fibroblasts transfected with cDNA for human IL-2 or IL-2Rβ genes. In L3 and Lαβ cells, transcription of the endogenous IL-2 gene was suppressed by cyclosporin A and enhanced by cycloheximide. These data may have implications for gene therapy of cancer cells.