Much attention has recently focused upon hepatocyte growth factor (HGF) as a potential regulator of epithelial branching morphogenesis. However, since neither the HGF nor c-met "knockout" mice show abnormal kidney branching morphogenesis, we sought to analyze the relative importance of HGF in in vitro branching morphogenesis compared with other factors secreted by the embryonic kidney. Exploiting an assay that employs kidney epithelial cells (murine inner medullary collecting duct, mIMCD3) seeded in collagen cocultured with the embryonic kidney, we found that a tyrosine kinase inhibitor that is highly specific for the epidermal growth factor (EGF) receptor (EGFR), tyrphostin AG1478, inhibited mIMCD3 cell process formation (an early step in branching tubulogenesis) by 40%, whereas high concentrations of neutralizing anti-HGF antibodies had a lesser effect (20% inhibition), suggesting that EGFR ligands account for a larger fraction of branching morphogens secreted by the embryonic kidney than HGF. In addition, when an embryonic epithelial cell line derived from c-met (-/-) mice was cocultured with the embryonic kidney, these c-met (-/-) cells underwent process formation. EGFR ligands but not HGF were able to induce branching tubulogenesis in these cells. All EGFR ligands tested, including EGF, transforming growth factor-alpha, heparin-binding EGF, betacellulin, and amphiregulin, induced mIMCD3 cell tubulogenesis. EGFR ligands caused upregulation of urokinase, urokinase receptor, and matrix metalloprotease-1, and tubulogenesis could be inhibited by the metalloprotease inhibitor 1,10-phenanthroline. Our results support the notion that multiple parallel and potentially redundant growth factor-dependent pathways regulate branching tubulogenesis.