The thymus leukemia (TL) antigens, encoded by class I genes in the Tla subregion of the major histocompatibility complex (MHC), are cell surface molecules expressed on thymocytes of certain strains of mice and on certain T-cell leukemias. In order to study the fine structure and interrelationships of genes of the Tla subregion, a Tla-specific probe was isolated from the TL-encoding T13c gene of BALB/c mice (Tlac haplotype). The probe hybridized with two Tla genes in the Tlac haplotype (T13c and T3c) and with only one in the Tlab haplotype (T3b). Examination of this subset of Tla genes (T3b, T3c, and T13c) by restriction enzyme analysis and oligonucleotide hybridization studies confirmed that T3b is the allele of T3c and that T3c and T13c may have arisen by duplication. The T3b gene, while not transcribed in the tissues of the TL- strain C57BL6, was shown to be transcriptionally active in the TL-expressing leukemic cell line ERLD derived from that strain. The T3b gene was cloned and its complete DNA sequence was determined. These data permit complete comparison of two Tla-region genes, T3b and its homologue T13c, and allow us to conclude that these genes show extraordinarily high sequence conservation, in contrast to alleles of the H-2K- and H-2D-region genes. Comparison of T3b with other class I sequences in the H-2 and Qa subregions suggests that the T3-subset genes are the most divergent from other class I genes.