Escherichia coli O157:H7 is a commensal organism in cattle, but it is a pathogen in humans. This differential expression of virulence suggests that specific virulence factors are regulated differently in human and bovine hosts. To test this hypothesis, relative real-time reverse transcription-PCR was used to relate the expression of several putative virulence genes (eae, espA, stx 2 , rfbE, ehxA, and iha) to that of the "housekeeping" gene gnd during natural human and experimental bovine infection with E. coli O157:H7. We examined these genes in fecal samples from eight humans and four calves. iha and espA were significantly more expressed in bovine infections. rfbE and ehxA appeared to be more highly expressed in human infections, though these differences did not achieve statistical significance. Our results support the hypothesis that some virulence-associated genes of O157:H7 are differentially expressed in a host-specific manner.Escherichia coli O157:H7, a human pathogen that causes diarrhea, bloody diarrhea, and the hemolytic uremic syndrome (HUS), has acquired multiple virulence factors that might aid its pathogenicity. Principal among these are the phage-encoded Shiga toxins (Stx proteins), which inhibit protein synthesis in eukaryotic cells (41), and the LEE pathogenicity island, which encodes products associated with formation of attaching-and-effacing lesions at the host surface (reviewed in reference 13). E. coli O157:H7 also has a 92-kb nonconjugative plasmid that encodes several putative virulence factors, including enterohemolysin (encoded by ehxA, also termed hlyA [1,5]), a member of the RTX family of exoproteins. E. coli O157:H7 also expresses the IrgA homologue adhesin (Iha), which was recently identified as a virulence factor in uropathogenic E. coli (16,38).Some of these factors can affect animal hosts. For example, E. coli O157:H7 causes attaching-and-effacing lesions on cultured bovine cells (31) and in neonatal calves (8). Despite this, E. coli O157:H7 is not known to cause natural disease in any host except humans. This discrepancy suggests the hypothesis that specific virulence factors of E. coli O157:H7 are regulated differently when the bacteria are in the human rather than the bovine gut. To test this hypothesis, real-time reverse transcription-PCR (RT-PCR) was employed to examine gene expression in fecal samples from children infected with E. coli O157:H7 and from experimentally infected calves. The resulting data support the hypothesis that virulence genes are differentially expressed in the human and bovine hosts.