In this study we demonstrate that human T cell receptor (TcR) chains can be co-expressed with murine CD3 on the cell surface of a murine T cell hybridoma. Human TcR alpha and beta genes from the Jurkat leukaemic cell line were transfected into a TcR-negative mouse T cell hybridoma, TG40. The Jurkat TcR was successfully co-expressed at the cell surface with mouse CD3 components. Brightly staining transfectants were selected by fluorescence-activated cell sorting, and levels of expression comparable to a normal T cell line were achieved suggesting that the human TcR dimer assembled efficiently with the mouse CD3 complex. Northern blot analysis demonstrated similar levels of TcR messenger RNA to those found in the parental Jurkat line. Although we have not formally demonstrated surface expression of the Jurkat TcR alpha chain, the residual alpha gene transcript which is present in murine TG40 line is non-expressible. In order to test the signalling capacity of this human/mouse complex, the transfectants were stimulated with an anti-V beta 8 monoclonal antibody. This stimulus led to interleukin-2 production by the transfectants, demonstrating the delivery of a transmembrane signal. In addition, B10.A mice were immunised with the transfectants, and the antisera from these mice stained the transfectant and the Jurkat cell line, but not the parental T cell hybridoma. This interspecies transfection approach should now permit us to explore the requirements for T cell activation, the constraints on TcR alpha beta chain pairing, and creates ideal reagents for inducing a mouse anti-human TcR-specific response with a view to producing panels of anti-human TcR monoclonal antibodies.