Avian homologues of mammalian gamma delta and alpha beta T-cell antigen receptors, TCR1 and TCR2, have been identified with monoclonal antibodies. These TCR isotypes are associated with the avian CD3 proteins on the T-cell surface. During chick development, T-cell subpopulations bearing CD3/TCR1 or CD3/TCR2 receptor complexes are generated sequentially in the thymus and seeded to the periphery in the same order. In this study, we used two-color immunofluorescence to identify the subsequent development of a third subpopulation of T cells, provisionally named TCR3. These CD3+ cells, which expressed neither TCR1 nor TCR2, were first detected in the blood 1 week after hatching and increased numerically as a function of age to account for approximately 15% of the circulating T-cell pool in adults. Most (greater than 80%) of the TCR3 cells expressed the CD4 accessory molecule. The relative incidence of the TCR3 subpopulation increased dramatically as a consequence of embryonic treatment with anti-TCR2 antibody and thymectomy after hatching. Two disulfide-linked polypeptides, of Mr 48,000 and 40,000, were associated with the CD3 complex on the TCR3 cells. Examination of the TCR protein backbones and peptide mapping of the TCR chains after partial proteolysis indicated that the TCR3 heterodimer differs from both TCR1 and TCR2. These results suggest the existence of a third class of T-cell receptors in birds.