We have studied the genetic diversity of the TCR repertoire to the murine alloantigen I-Abm12 by generating a panel of 178 C57BL/10-derived I-Abm12-reactive T cell hybridomas. The expression of V alpha and V beta gene families was examined in this panel and the frequency of expression of V beta, but not ofV alpha, gene families differed significantly from that observed in a companion panel of random C57BL/10-derived hybridomas. The V beta 5 gene family was expressed significantly less frequently while the V beta 14, V beta 15, and V beta 16 genes were expressed significantly more frequently in the panel of I-Abm12-reactive than in the panel of random hybridomas. The junctional regions (VJ alpha and VDJ beta) of TCR V alpha and V beta genes from selected I-Abm12-specific hybridomas were amplified using the polymerase chain reaction, and directly sequenced. Surprisingly, no conserved J alpha, D beta, J beta, or N region-encoded sequences among these selected I-Abm12-reactive TCRs were identified. Thus, the T cell response to an I-A alloantigen that differs by only three amino acid residues from the I-A molecule of the responding strain is genetically complex but nonrandom. We have estimated that the repertoire to this alloantigen is comprised of at least 37 different TCRs.