A considerable fraction of T cells express two distinct T cell receptors (TCR), mainly due to expression of two TCR alpha chains. It has been suggested that such dual-TCR cells could have a role in autoimmunity. However, as such cells express less of each TCR, they could be less sensitive to their physiological ligand, i.e. peptide plus major histocompatibility complex molecules (MHC). We tested this hypothesis in a transgenic TCR model in which most T cells express different amounts of the transgene-encoded TCR, due to expression of endogenous TCR alpha chains. Five Th1 clones derived from lambda2(315) immunoglobulin light chain-specific TCR-transgenic mice expressed different levels of the transgene-encoded TCR, ranging from approximately 10,000 to approximately 50,000 TCR per cell. Cytosolic Ca2+ mobilization in single T cells from these clones elicited by lambda2(315) peptide-pulsed, I-Ed-expressing antigen-presenting cells, correlated linearly with the relative transgene-encoded TCR expression. The peptide requirement for half-maximal T cell proliferation showed a similar correlation, with low TCR levels requiring higher peptide concentration. Corroborative evidence was obtained by deployment of short-term polyclonal CD4+ lines from TCR-transgenic mice. Such lines had reduced early (Ca2+ mobilization) and late (lymphokine and proliferation) responses, compared with T cell lines from recombination-deficient TCR-transgenic severe combined immunodeficiency mice (which express only a single transgene-encoded TCR). Taken together, the Ca2+ responses increase gradually with increasing TCR expression per cell, similar to the previously described analog Ca2+ signaling elicited by increasing amounts of peptide/MHC [Røtnes et al., Eur. J. Immunol. 1994. 24: 851]. Surprisingly small reductions in TCR expression per cell reduce T cell responsiveness. This suggests that dual-TCR T cells are immunologically less effective than single-TCR T cells.