The nucleotide sequence of a tRNALeuCUG gene from Drosophila melanogaster has been determined and compared with available tRNALeuCUG sequences from other eukaryotes, as well as with the tRNALeuUUG gene of D. melanogaster. The genomic location, determined by in situ hybridization, was found to be at site 66B on chromosome 3L. This localization probably places it within one of the known, but uncharacterized, clusters of tRNA genes in this organism. In addition, the transcriptional behaviour of this tRNALeuCUG gene in various in vitro systems is described and it seems that, although the gene is transcribed in all test systems, the very A + T-rich 5'-flanking sequence of this particular gene may be somewhat inhibitory to transcription in vitro.