At least 13 independent tRNAVal gene loci were detected in the human genome. Three of these genes were isolated and shown to occur only once in the haploid genome. No further functional tRNA genes are located on the isolated clones. Two tRNAVal genes encode the known major and minor tRNAVal isoacceptors, the third may be a pseudogene because a corresponding tRNAVal is not yet known. Comparison of extragenic sequences did not reveal significant homologies, indicating the separation of these genes early in vertebrate evolution. An Alu-type repeat was found in two of the clones within several hundred bp distance from the tDNA. All three genes are transcriptionally active in a HeLa nuclear extract. We show here for the first time that homologous in vitro transcription of mammalian tRNA genes strongly depends on extragenic control regions: interestingly, as a consequence of different flanking regions, the transcription efficiencies vary by an order of magnitude among the genes for the major and the minor tRNAVal and thus reflect the concentrations of these tRNAs in vivo.