Embryonic stem (ES) cells are a powerful tool for developmental biology and have been shown to respond to epidermal growth factor (EGF). However, little is known about the effect of EGF on the proliferation of ES cells, although many cellular functions are tightly regulated by EGF. Therefore, we examined the effect of EGF on the proliferation of mouse ES cells and their related signal pathways. EGF increased [³H] thymidine and BrdU incorporation in a time- and dose-dependent manner. EGF stimulated the phosphorylation of EGF receptor (EGFR). In the present study, inhibition of EGFR tyrosine kinase using AG 1478 or herbimycin A, inhibition of phospholipase C using neomycin or U 73122, inhibition of protein kinase C using bisindolylmaleimide I or staurosporine, and inhibition of L-type Ca²⁺ channel using nifedifine or methyoxyverapamil prevented EGF-induced [³H] thymidine incorporation. PKC ,_I,,,and were translocated to the membrane in response to EGF. EGF also increased [Ca²⁺]_i, which was blocked by the extracellular Ca²⁺ chelator, EGTA and the intracellular Ca²⁺ chelator, BAPTA-AM. EGF-induced increase of [Ca²⁺]_i was attenuated by inhibition of the Ca²⁺ channel. Moreover, inhibition of EGFR tyrosine kinase, PLC, and PKC completely prevented EGF-induced increases in [Ca²⁺]_i. Inositol phosphates levels were also increased by EGF and these increases were blocked by EGFR tyrosine kinase inhibitors. Furthermore, EGF rapidly increased formation of H₂O₂, and the pretreatment of antioxidant (NAC) inhibited EGF-induced increase of [Ca²⁺]_i. In addition, we observed p44/42 MAPKs phosphorylation, but not p38 MAPK phosphorylation by EGF. Inhibition of EGFR tyrosine kinase, PLC, PKC, or Ca²⁺ channel blocked EGF-induced phosphorylation of p44/42 MAPKs. Inhibition of p44/42 MAPKs using PD 98059 (MEK inhibitor) attenuated EGF-induced increase of [³H] thymidine incorporation, but not SB 203580 (p38 MAPK inhibitor). Finally, inhibition of EGFR tyrosine kinase, PKC, Ca²⁺ channel, or p44/42 MAPKs attenuated EGF-stimulated cyclin D1, cyclin E, cyclin-dependent kinase 2 (CDK 2), and CDK 4, respectively. In conclusion, EGF partially stimulated proliferation of mouse ES cells via PLC/PKC, Ca²⁺ influx, and p44/42 MAPKs signal pathways through EGF receptor tyrosine kinase phosphorylation.