Two nonallelic human tRNAiMet genes were assigned to chromosome 6 by filter hybridization of DNA from human-rodent somatic cell hybrids by using probes containing unique sequences from the regions flanking each tRNAiMet gene. These unique sequence probes thus allowed each tRNAiMet gene to be analyzed individually in cell hybrids. Both tRNAiMet genes segregated in the hybrid cells with the chromosome 6 enzyme markers, soluble malic enzyme and the mitochondrial form of superoxide dismutase, and also with a karyotypically normal chromosome 6. By using hybrid clones containing translocations that divide chromosome 6 into five segments, both tRNAiMet genes were assigned to the p23 leads to q12 region. These results raise the possibility that other tRNAiMet genes may be syntenic with the two described in this study and illustrate the utility of using unique flanking sequences to identify members of a multigene family.