A subgroup of benign pleomorphic adenomas of the salivary glands is characterized by translocations, or on rare occasions deletions, with breakpoints at 3p21. We have applied restriction fragment length polymorphism (RFLP) analysis to assess the frequency of allelic losses at four different loci located within 3p21-->p25 in 35 pleomorphic adenomas, 18 of which were also karyotyped. Parallel analysis of constitutional and tumor DNAs in informative tumors revealed that all patients retained heterozygosity in their tumor DNA at the D3S2 and RAF1 loci. Among the 29 tumors informative for THRB three showed loss of heterozygosity (LOH). All three tumors had a t(3;8)(p21;q12). Of the 23 tumors informative for D3F15S2, one showed LOH. This tumor also had a t(3;8)(p21;q12). To further map the deletions in relation to the 3p21 translocation breakpoint, we also sublocalized the THRB locus. Using in situ hybridization we assigned the gene to 3p24.1-3. The fact that none of the tumors with loss of 3p alleles showed cytogenetic evidence of deletions indicates that the losses are submicroscopic, probably interstitial, and in most cases distal to the 3p21 breakpoint. This was confirmed in one case with loss of a THRB allele where both proximal (D3F15S2) and distal (RAF1) markers retained heterozygosity. Our results suggest that deletion of 3p sequences might be of progressional importance in a subset of pleomorphic adenomas with t(3;8)(p21;q12).