T cell antigen receptor (TcR) heterodimers of both the Ti-alpha beta and Ti-gamma delta types are expressed at the surface of T cells noncovalently associated with the CD3 complex composed of the monomorphic chains gamma, delta, epsilon and zeta. The structural relationship and assembly of the various components of this multimeric protein complex is still not fully understood. In this report, the human leukemic T cell line Lyon which expresses a Ti-gamma delta/CD3 complex, was characterized and compared to another human leukemic T cell line Jurkat (Ti-alpha beta/CD3). Membrane TCR-/CD3- variants of the T cell Lyon were induced and found to produce all of the Ti/CD3 components, with the exception of Ti-delta. Biochemical analysis indicated that: (1) Ti-gamma/CD3 gamma, delta, epsilon complexes were formed in the endoplasmic reticulum in the absence of Ti-delta; (2) the CD3-zeta chain did not associate with the Ti-gamma/CD3 gamma delta epsilon complex and (3) the Ti-delta chain was required for cell surface expression of the Ti-gamma delta/CD3 complex. Introduction of Jurkat wild-type Ti-alpha cDNA into Lyon T cells resulted in Ti-alpha beta/CD3 expression and abrogated Ti-gamma delta/CD3 expression. In contrast, the expression of the Ti-gamma delta/CD3 complex was not affected by transfection of a mutated Ti-alpha cDNA into Lyon cells. The mutated Ti-alpha chain formed complexes with Ti-beta and CD3 gamma delta epsilon, but the CD3-zeta chain did not associate with these complexes. Taken together analysis of Lyon cells transfected with either wild-type or mutated Ti-alpha suggested that the CD3-zeta chain may have higher affinity for Ti-alpha beta/CD3 complexes than for Ti-gamma delta/CD3 complexes.