The cloned murine cell line BK-BI-2.6.C6 has previously been shown to exhibit T cell characteristics, to synthesize and express MHC class II molecules, and to present protein antigens to antigen-dependent T cell clones. As a more definitive proof of the T-cell nature of these cells, transcripts of the rearranged T cell antigen receptor (TcR) beta gene were assessed by Northern blot analysis. BK-BI-2.6.C6 cells constitutively transcribe mRNA for the light chain of TcR and express the disulphide-linked alpha, beta TcR heterodimer at the cell surface. In addition mRNA for the polymorphic MHC class II subunits A alpha and A beta as well as for the invariant gamma chain were detected. BK-BI-2.6.C6 T cells effectively stimulated bovine insulin-reactive T hybridoma cells to lymphokine production in the presence of this antigen. Since the antigen-presenting and the responding T cell populations are maintained in culture in the absence of feeder cells, contamination by conventional accessory cells is excluded. These data unequivocally demonstrate that cloned murine Ia-expressing T cells can act as antigen-presenting accessory cells.