About 12 tRNAimet genes have been found at scattered locations in the human genome. Four fragments of human fetal liver DNA ranging in size from 11 to 18 kb, each containing a single tRNAimet gene, were cloned from a recombinant phage library. On the basis of restriction site mapping, electron microscopic analysis of heteroduplex structures and the maps of sequences transcribed in vivo in human fibroblasts, obtained by a novel contract-hybridization method, the fragments were shown to represent two different loci with homologies limited to several dispersed repetitive sequences within each of the chromosomal neighborhoods. Detailed structural analysis of the tRNA regions revealed several blocks of homology at the proximal flanking sequences of the two nonallelic genes, one of which differed from the common vertebrate tRNAimet sequence by a base substitution at position 56 with a T in place of G. Two oligonucleotides identical or very similar to tRNAimet structural sequences were present at the 5' border of both genes. A review of published sequence data showed other unequivocal examples of tRNA-coding sequences present at the 5' flanking region of the associated tRNA gene, which in several cases contained the site of transcription initiation.