We have previously reported that human T cell receptors (TcR) selected in the class II-restricted (HLA-DRB1*1302) response to a tetanus toxin peptide (tt830-843) frequently used the V beta 2 germ-line segment which paired with several V alpha segments and that the putative CDR3 of both alpha and beta chains showed remarkable heterogeneity. To analyze the structural basis for recognition of the tt830-843/DR complex, five of these TcR were reconstituted into a murine T cell hybridoma, 58 alpha- beta-, by expressing the human alpha and beta variable regions joined to the mouse alpha and beta constant regions, respectively. The chimeric TcR, expressing the same V beta germ-line segment (V beta 2), two expressing V alpha 21.1, two V alpha 17.1 and one V alpha 8.1 were shown to have the expected antigen specificity and DR restriction. Two lines of evidence suggested that the putative CDR3, although not conserved in these TcR, played a key role in recognition. First, two TcR with identical V germ-line segments but distinct CDR3 showed large difference in their capacity to react with the ligand. Second, interchanging the alpha and beta chains from tt830-843/DR1302-specific TcR which differed in their CDR3 sequences invariably led to loss of recognition. We also asked whether germ-line V alpha 17.1 could functionally replace V alpha 21.1, as they appear to be related in their primary sequence. However, as in the case of CDR3 exchanges, V alpha replacement abrogated TcR reactivity. Taken together, these data underline the fine interdependence of the structural components of the TcR binding site in defining a given specificity. Four of the TcR studied displaying promiscuous recognition were also tested against different DR alleles and site-directed mutants. The results of these experiments suggested that, in spite of their structural heterogeneity, anti-tt830-843 TcR may have a similar orientation with respect to the peptide/DR complex. The reconstitution system described herein should represent a valuable tool for detailed studies of human TcR specificity.