Two human tRNA genes were isolated from a human genomic DNA library and their sequences have been determined. The human-lambda recombinant DNA library has been screened using a purified human tRNA(Ser) and 5 different clones have been identified. A 3.1 Kb EcoRI DNA fragment, named lambda St-19A which was originated from one of these clones, was subcloned into pUC 9 vector plasmid for further analyses. Within DNA sequences of lambda St-19A, two different human tRNA(Ser) genes were found on opposite DNA strands with a gap of 2.5 Kb ; one, a tRNA gene(Ser, AGA) which is identical in sequence to a previously reported human tRNA(Ser, AGA) gene from lambda St-1B; the other, a tRNA(Ser, TGA) genes. The great important thing was found by the examination of flanking sequences of t-RNA genes. The 110 base pairs of 5'-flanking region and 82 base pairs of tRNA coding region from lambda St-19A are exactly same as those from lambda St-1B. But the 3'-flanking region is different from each other. These results demonstrate that one of these tRNA(Ser) genes has arisen through a duplication event.