The calcium-sensing receptor (CaR) is a G-protein-coupled receptor that is activated by extracellular calcium (Cao2+). Rat-1 fibroblasts have been shown to proliferate and increase ERK activity in response to elevation of [Ca2+]o, and these responses are dependent on functional CaR expression. In this report, we examined the role of cross-talk between the CaR and the epidermal growth factor receptor (EGFR) in mediating these responses in Rat-1 cells. This report shows that AG1478, a specific inhibitor of the EGFR kinase, significantly inhibits the increase in proliferation induced by elevated Cao2+. Furthermore, we show that AG1478 acts downstream or separately from G protein subunit activation of phospholipase C. AG1478 significantly inhibits Cao2+-stimulated ERK phosphorylation and in vitro kinase activity. A similar inhibition of ERK phosphorylation was observed in response to the inhibitor AG494. In addition, treatment with inhibitors of metalloproteases involved in shedding of membrane anchored EGF family ligands substantially inhibited the increase in ERK activation in response to elevated Cao2+. This is consistent with the known expression of TGFalpha by Rat-1 cells. These results indicate that EGFR transactivation is an important component of the CaR-mediated response to increased Cao2+ in Rat-1 fibroblasts and most likely involves CaR-mediated induction of regulated proteolysis and ligand shedding.