Epidermal growth factor (EGF) is a multifunctional factor known to influence proliferation and function of a variety of cells. The actions of EGF are mediated by EGF receptor tyrosine kinase pathways, including stimulation of phospholipase Cgamma and mobilization of intracellular Ca(2+) ([Ca(2+)](i)). Generally, agonist-mediated Ca(2+) mobilization involves both Ca(2+) release from internal stores and Ca(2+) influx activated by store depletion (i.e. capacitative or store-operated Ca(2+) influx). However, the role of capacitative Ca(2+) entry in EGF-mediated Ca(2+) mobilization is still largely unknown. In this study, we compared [Ca(2+)](i) signals elicited by EGF with those induced by agents (the muscarinic receptor agonist carbachol and thapsigargin (Tg)) known to activate capacitative Ca(2+) entry. Unlike carbachol and Tg, EGF (5 nm) elicited a transient [Ca(2+)](i) signal without a plateau phase in the presence of extracellular Ca(2+) and also failed to accelerate Mn(2+) entry. Repletion of extracellular Ca(2+) to cells stimulated with EGF in the absence of Ca(2+) elicited an increase in [Ca(2+)](i), indicating that EGF indeed stimulates Ca(2+) influx. However, the influx was activated at lower EGF concentrations than those required to stimulate Ca(2+) release. Interestingly, the phospholipase C inhibitor completely inhibited Ca(2+) release induced by both EGF and carbachol and also reduced Ca(2+) influx responsive to carbachol but had no effect on Ca(2+) influx induced by EGF. EGF-induced Ca(2+) influx was potentiated by low concentrations (<5 ng/ml) of oligomycin, a mitochondrial inhibitor that blocks capacitative Ca(2+) influx in other systems. Transient expression of the hTRPC3 protein enhanced Ca(2+) influx responsive to carbachol but did not increase EGF-activated Ca(2+) influx. Both EGF and carbachol depleted internal Ca(2+) stores. Our results demonstrate that EGF-induced Ca(2+) release from internal stores does not activate capacitative Ca(2+) influx. Rather, EGF stimulates Ca(2+) influx via a mechanism distinct from capacitative Ca(2+) influx induced by carbachol and Tg.