Epidermal growth factor (EGF) is linked to the pathogenesis of polycystic kidney disease (PKD). We explored signaling pathways activated by EGF in orpk cilia (-) collecting duct cell line derived from a mouse model of PKD (hypomorph of the Tg737/Ift88 gene) with severely stunted cilia, and in a control orpk cilia (+) cell line with normal cilia. RT-PCR demonstrated mRNAs for EGF receptor subunits ErbB1, ErbB2, ErbB3, ErbB4, and mRNAs for Na+/H+ exchangers (NHE), NHE-1, NHE-2, NHE-3, NHE-4, and NHE-5 in both cell lines. EGF stimulated proton efflux in both cell lines. This effect was significantly attenuated by MIA, 5-(n-methyl-N-isobutyl) amiloride, a selective inhibitor of NHE-1 and NHE-2, and orpk cilia (-) cells were more sensitive to MIA than control cells (P < 0.01). EGF significantly induced extracellular signal-regulated kinase (ERK) phosphorylation in both cilia (-) and cilia (-) cells (63.3 and 123.6%, respectively), but the effect was more pronounced in orpk cilia (-) cells (P < 0.01). MIA significantly attenuated EGF-induced ERK phosphorylation only in orpk cilia (-) cells (P < 0.01). EGF increased proliferation of orpk cilia (-) cells and orpk cilia (-) cells, respectively, and MIA at 1–5 μM attenuated EGF-induced proliferation in orpk cilia (-) cells without affecting proliferation of orpk cilia (+) cells. EGF-induced proliferation of both cell lines was significantly decreased by the EGFR tyrosine kinase inhibitor AG1478 and MEK inhibitor PD98059. These results suggest that EGF exerts mitogenic effects in the orpk cilia (-) cells via activation of growth-associated amiloridesensitive NHEs and ERK.