Adoptive transfer of genetically T-cell receptor (TCR)–modified lymphocytes has been recently reported to cause objective cancer regression. However, a major limitation to this approach is the mispairing of the introduced chains with the endogenous TCR subunits, which leads to reduced TCR surface expression and, subsequently, to their lower biological activity. We here show that it is possible to improve TCR gene transfer by adding a single cysteine on each receptor chain to promote the formation of an additional interchain disulfide bond. We show that cysteine-modified receptors were more highly expressed on the surface of human lymphocytes compared with their wild-type counterparts and able to mediate higher levels of cytokine secretion and specific lysis when cocultured with specific tumor cell lines. Furthermore, cysteine-modified receptors retained their enhanced function in CD4⁺ lymphocytes. We also show that this approach can be employed to enhance the function of humanized and native murine receptors in human cells. Preferential pairing of cysteine-modified receptor chains accounts for these observations, which could have significant implications for the improvement of TCR gene therapy. [Cancer Res 2007;67(8):3898–903]