Pathogenicity islands (PAls) contain virulence determinants and are often linked to tRNA loci. The leuX gene encoding tRNA5Leu is associated with PAI II536 of the uropathogenic E. coli strain 536 (O6:K15:H31) and is important for the expression of virulence factors in this strain. Transcription of leuX differs from that of the majority of tRNA genes including the major leucyl-tRNA gene leuV. Availability of the tRNAsLeu can be linked with the efficient expression of virulence determinants. Transcription of leuX and letV was studied to gain further insights into the regulation of this tRNA gene and its role during the expression of virulence determinants. leuX but not letV transcripts accumulated in stationary phase. While leuX transcription was unchanged or slightly up-regulated by an increase in growth temperature, osmolarity or ethanol concentration, that of leuV was reduced. The heat shock-specific sigma factor RpoH was shown to affect transcription of leuX but not of leuV. These results demonstrate that transcription of a tRNA gene can be specifically influenced by an alternative sigma factor. Our finding expands our knowledge of the regulation of tRNA gene transcription and represents an additional mode of regulation of gene expression that can also affect bacterial virulence.