The structure of the DRB1*03 gene has been interpreted as the product of a gene conversion event involving a DRB3 gene as donor and resulting in the introduction of two short segments of the DRB3 sequence into the DRB1 locus. The serological counterpart of this double insertion is the TR81 specificity. Consequently, the TR81-specifying sequences can reside on either DRB1 or DRB3, or on both loci. Within each of the two sequence stretches a single nucleotide may be responsible for the generation of the TR81 alloantigen. Oligonucleotide probes corresponding to these stretches and to their allelic variants were constructed. They were used, under stringent hybridization conditions, to detect TR81-specifying sequences in the DNA of HLA-homozygous cell lines carrying different haplotypes of the DRw52 family. Prior to hybridization the DNA was amplified with either DRB1-specific or DRB3-specific primers. Using this approach it was possible to perform a "DNA typing" of the TR81-specifying sites separately on both the DRB1 locus and the DRB3 locus.