The gut origin hypothesis for sepsis implies that the gastrointestinal (GI) tract of human is the source of septicaemic events in patients due to bacterial translocation from this site. The urinary tract is also an important site for translocation and has been linked to the passage of pathogenic Escherichia coli into the blood stream via the kidney, a process known as urosepsis. The aim of this study was to investigate whether E. coli isolated from the blood of patients with urinary tract infection (UTI) have the ability to translocate from the GI tract of the same patient while they are also causing UTI. A collection of 47 pairs of E. coli isolated from the blood and urine of hospitalised patients with urosepsis were investigated for their ability to adhere and translocate in cell lines representing renal cells (A-498) and GI epithelial cells (HT-29). An initial comparison of the strains isolated from urine and blood showed that 44 out of 47 (94%) patients had strains that were identical. Blood isolates adhered to both cell lines although their rate of adhesion to A-498 cells was significantly higher than those of HT-29 cells (5.8 ± 3.8 per cell versus 2.8 ± 1.9; P < 0.0001). The rate of translocation in A-498 cells was also significantly (P < 0.0001) higher after 120 minutes (8.6 x 105 versus 3.0 x 105). The three non-identical blood isolates were unable to translocate HT-29 cells, indicating that host immune factors might be more important than bacterial ability to translocate the GI epithelium in these patients. We suggest that in hospitalised patients with UTI and septicaemia E. coli may translocate from other sources such as the GI tract, hence questioning the assumption that the urinary tract is the only source of septicaemia in these patients.