We report the isolation and characterization of a 31 X 10(3) base-pair DNA segment containing a cluster of Drosophila melanogaster transfer RNA genes from the cytogenic locus 90BC. Seven distinct coding regions have been identified in a 15 X 10(3) base-pair DNA segment. These coding regions contain at least ten tRNA structural genes and include sequences encoding the following tRNAs: tRNAval, tRNAPro, tRNAAla and tRNAThr. We have determined the nucleotide sequence of six of these structural genes and their flanking regions. These genes do not contain intervening sequences nor do they encode the terminal CCA. The tRNA genes from the locus also appear to be functional when assayed in a Xenopus germinal vesicle in vitro transcriptional system.