Enteropathogenic Escherichia coli (EPEC) have been previously shown to alter sodium hydrogen exchanger 3 (NHE3) activity in human intestinal epithelial cells. To further characterize these observations, PS120 fibroblasts transfected with NHE3 were studied. EPEC E2348/69 infection decreased NHE3 activity in PS120 fibroblasts. The effect on NHE3 was enhanced when PS120 cells were co-transfected with the scaffolding/regulatory proteins NHERF1 or NHERF2 or EBP50 and E3KARP respectively. The decrease in NHE3 activity was dependent on an intact type III secretion system, although intimate attachment mediated by translocated intimin receptor was not required. Despite its ability to bind to NHERF proteins, the EPEC effector Map had no impact on the regulation of NHE activity. Instead, EspF was found to be responsible for decreased NHE3 activity. However, neither EspF-induced apoptosis nor the interaction of EspF with sorting nexin-9, an endocytic protein, were involved.