The coordinate expression of a marker gene and a therapeutic gene in one retroviral vector has considerable advantages. High-titer producer lines can potentially be selected on the basis of marker gene expression, and the expression of transduced genes in target cells can readily be followed. Moreover, target cells with stable high expression can be selected before use in therapeutic protocols or research questions. We used internal ribosomal entry site (IRES) sequences to express two genes in the same retroviral vector. We used the LacZ gene as the marker gene and the cytokine interleukin (IL)-7 or dominant negative (dn) forms of the T-cell tyrosine kinases ZAP-70 and lck as genes of interest. Amphotropic packaging cells transfected with MFG-IL-7-IRES-LacZ, MFG-dnZAP-70-IRES-LacZ, or MFG-dnlck-IRES-LacZ were sorted on the basis of beta-galactosidase expression. These LacZ-positive producer cells also expressed the gene of interest, produced high-titer retrovirus, and were capable of efficiently transducing Jurkat T cells and T-cell clones. When MFG-IL-7-IRES-LacZ-transduced Jurkat T cells were sorted on the basis of LacZ expression, a positive correlation with the amount of IL-7 produced by these cells was found. This demonstrates that selection of the LacZ marker gene also selects for cells that express the gene of interest at high levels. Moreover, T cells transduced with the dn tyrosine kinases and selected on the basis of LacZ expression showed functional alterations after T-cell receptor stimulation, demonstrating that retrovirally transduced signaling molecules can alter the function of T cells.