Background: T cell receptor gene transfer is a promising strategy to treat patients suffering from HPV induced malignancies. Therefore we isolated the TCRαβ open reading frames of an HPV16E6 specific CTL clone and generated TCR transgenic T cells. In general low level expression of the transgenic TCR in recipient human T cells is observed as well as the formation of mixed TCRs dimers. Here we addressed both issues employing three different expression platforms.Methods: We isolated the HVP16E6 specific TCRα and TCRβ open reading frames and retrovirally transduced human T cells with either wild-type (wt), or codon-modified (cm) chains to achieve enhanced TCR expression levels, or used codon-modification in combination with cysteinization (cmCys) of TCRs to facilitate preferential pairing of the introduced TCRα and TCRβ chains.Results: Careful analysis of recipient T cells carrying the HPV16E6 TCRβ and endogenous TCR chains revealed the transgenic TCRβ chain to behave very promiscuously. Further analysis showed that the percentage of tetramer positive T cells in codon-modified/cysteinized TCR transgenic T cells was four-fold higher compared to wild-type and two-fold higher compared to codon-modification only. Functional activity, as determined by IFN-γ production, was high in cmCysTCR transgenic T cells, where it was low in cm and wt TCR transgenic T cells. Recognition of endogenously processed HPV16E6 antigen by cmCysTCR transgenic T cells was confirmed in a cytotoxicity assay.Conclusions: Promiscuous behavior of the HPV16E6 specific TCRβ chain can in part be forced back into specific action in TCR transgenic T cells by codon modification in combination with the inclusion of an extra cysteine in the TCR chains.