A subgroup of pleomorphic adenomas of the salivary glands is characterized by translocations involving chromosome 8, with consistent breakpoints at 8q12. As part of a positional cloning effort to isolate the gene(s) affected by these translocations we now report the mapping of the 8q12 breakpoint in two primary pleomorphic adenomas with the recurrent t(3;8)(p21;q12). Yeast artificial chromosome (YAC) clones corresponding to eight different loci in 8q11-12 were isolated and mapped by fluorescence in situ hybridization (FISH). The t(3;8) breakpoint was mapped within a 1 Mb region flanked by MOS proximally and by the genetic marker D8S166 distally. One YAC within this region was shown to span the t(3;8) breakpoint in two tumors. This YAC will provide an excellent tool for isolating the gene(s) at the breakpoint(s) in adenomas with t(3;8).