Enteropathogenic Escherichia coli (EPEC) increases sodium/hydrogen exchanger 2 (NHE2)-mediated sodium uptake by intestinal epithelial cells in a type III secretion-dependent manner. However, the mechanism(s) underlying these changes are not known. This study examines the role of a number of known secreted effector molecules and bacterial adhesins as well as the signaling pathways involved in this process. Deletion of the bacterial adhesins Tir and intimin had no effect on the increase in sodium/hydrogen exchanger (NHE) activity promoted by EPEC infection; however, there was a significant decrease upon deletion of the bundle-forming pili. Bacterial supernatant also failed to alter NHE activity, suggesting that direct interaction with bacteria is necessary. Analysis of the signal transduction cascades responsible for the increased NHE2 activity during EPEC infection showed that PLC increased Ca2+, as well as PKCalpha and PKCepsilon were involved in increasing NHE activity. The activation of PKCepsilon by EPEC has not been previously described nor has its role in regulating NHE2 activity. Because EPEC markedly increases NHE2 activity, this pathogen provides an exceptional opportunity to improve our understanding of this less-characterized NHE isoform.