Although T-cell receptor (TCR) transgenic as well as knockout and knockin mice have had a large impact on our understanding of T-cell development, signal transduction and function, the need to cross these mice delays experiments considerably. Here we provide a methodology for the rapid expression of TCRs in mice using 2A peptide-linked multicistronic retroviral vectors to transduce stem cells of any background before adoptive transfer into RAG-1(-/-) mice. For simplicity, we refer to these as retrogenic mice. We demonstrate that these retrogenic mice are comparable to transgenic mice expressing three commonly used TCRs (OT-I, OT-II [corrected] and AND). We also show that retrogenic mice expressing male antigen-specific TCRs (HY, MataHari and Marilyn) facilitated the analysis of positive and negative selection in female and male mice, respectively. We examined various tolerance mechanisms in epitope-coupled TCR retrogenic mice. This powerful resource could expedite the identification of proteins involved in T-cell development and function.