We have sequenced 22 TCR-alpha clones isolated from two cDNA libraries derived from rat thymocytes and heart allograft-infiltrating lymphocytes. Sixteen different V alpha and 17 J alpha genes were characterized in these clones. The V alpha genes could be divided into nine subfamilies, each containing from one to three members. Nucleotide sequence comparisons to mouse alpha genes revealed the existence of mouse homologues for all the J alpha s and 8 of 9 V alpha subfamilies. One of the rat V alpha subfamilies appeared to have no known mouse counterpart. Southern blot analysis of germ-line DNA with cDNA probes specific for six different subfamilies demonstrated the existence of two to nine V alpha genes per subfamily. A minimum number of 35 V alpha genes was estimated for the nine rat subfamilies characterized. These analyses also detected two different RFLP patterns among five different inbred rat strains, including strain-specific loss of hybridization bands for some V alpha, indicating that deletion events may have occurred in the rat alpha gene locus. The characterization of rat TCR V alpha genes should facilitate a better understanding of T cell role in pathologic conditions such as autoimmune diseases and graft rejection.