Virulence gene expression in enteropathogenic Escherichia coli (EPEC) is governed by a combination of environmental factors and virulence regulators. These factors control the expression of the bundle-forming pili (BFP), intimin, the type III secretion apparatus and the secreted proteins EspA, EspB, EspD and Tir. Expression of the bfp genes occurs for a short period in early exponential phase during growth in tissue culture medium. The nucleoid-associated regulator protein, Fis, is also expressed transiently during this period. To determine whether Fis was responsible for the growth phase-dependent expression of bfp, fis was deleted from the EPEC strain E2348/69S. Paradoxically, the Delta fis mutant retained the ability to colonize HEp-2 cells in a characteristic localized adherence pattern, and Fis was found negatively to regulate the expression of BFP. However, the Delta fis mutant failed to induce the accretion of filamentous actin, which is associated with attaching and effacing lesions. Using a combination of Western blotting and a novel multiplex primer extension assay (MPEA), we showed that, although the expression of intimin and Tir was not affected, transcription of the LEE4 operon encoding espADB and the virulence activator, Ler, were found to be Fis dependent.