The uropathogenic Escherichia coli strain 536 (O6:K15:H31) carries two α-haemolysin determinants which are located on different pathogenicity islands (PAI I536 and PAI II536). PAI II536 is associated with the tRNA gene leuX. The leuX-encoded tRNA5Leu is required for the efficient expression of the hly determinants in strain 536. HlyA levels were reduced and secretion of the protein was delayed in the leuX-negative mutant strain 536Δ102. The lack of a functional tRNA5Leu resulted in a decrease in hly transcript levels in comparison to the wild-type strain. Analysis of several genes whose products are involved in the regulation of hly expression revealed that levels of RfaH and Hha, as well as the corresponding rfaH and hha transcripts, were higher in the leuX-negative background, whereas the expression of tolC and hns was not influenced by the leuX genotype. The analysis of hly transcript levels in hha deletion mutants of the E. coli strains 536 and 536Δ102 demonstrated that the increase in hha expression is partially responsible for the reduction in hly transcript levels in the leuX-negative background. These results demonstrate that the tRNA5Leu affects the expression of the α-haemolysin determinant at different levels in a regulatory cascade, and imply that, in addition to Hha, at least one further, as yet unidentified, regulatory factor must be involved in the regulation of hly transcription in the uropathogenic E. coli strain 536.