We have constructed a specifically primed thymus cDNA library to study the V-region repertoire of the TCR beta-chain in the laboratory rat. From this library, we have isolated and sequenced 14 clones that hybridize with beta-chain constant region probes, eight of which contain V gene segments. Two of these eight contain a V gene segment whose sequence is 85% identical to mouse V beta 14, which has been estimated to be present in mouse thymocyte cDNA at a frequency of only 1.2%. Of the remaining six, four appear to be homologues of known mouse V beta sequences, and average 88% identity with their mouse counterparts. Two additional rat V regions have no homologue yet identified in the mouse, showing only 64 and 73% identity with the most closely related mouse sequences; at least one of these is present and polymorphic in the mouse genome, and may be located near one end of the V beta deletion known in the SWR strain. The availability and pattern of V beta gene segment expression in rat thymus appears, therefore, to differ significantly from the mouse. Two cDNA clones represent germline transcripts which show a J/C splice (one of which is defective) but no D/J rearrangement; two different J segments are used, indicating the presence of promoter activity 5' to these two J segments. In addition, by using V beta-specific probes derived from our cDNA clones, we have analyzed the distribution of V beta alleles among 10 inbred strains of rat. We find that rat V beta gene segments occur predominantly in single member subfamilies, and present evidence for the existence of three TCR V beta haplotypes defined by RFLP.