To directly define the role of phospholipase Cgamma1 (PLCgamma1) in NF-kappaB activation, NF-kappaB promoted luciferase reporter gene plasmid (pNF-kappaB-Luc) was transfected into rat-3Y1 fibroblasts that overexpress whole PLCgamma1 (PLCgamma1-3Y1), src homology domains SH2-SH2-SH3 of PLCgamma1 (SH223-3Y1) and v-src (Src-3Y1). Transient transfection with pNF-kappaB-Luc remarkably increased the luciferase activity in all three transformants compared with normal rat-3Y1 cells. Pretreatment with inhibitors of protein tyrosine kinase reduced this increase in luciferase activity, but U73122 (a PLC inhibitor) did not. While PD98059, an inhibitor of mitogen activated protein kinase (MAPK), significantly reduced the luciferase activity, there was no effect by wortmannin and Ro-31-8220, inhibitors of phosphatidylinositol 3-kinase (PI3K) and protein kinase C (PKC), respectively. This study shows a direct evidence that the SH2-SH2-SH3 region of PLCgamma1 contributes to the NF-kappaB signaling and that MAPK, but not PI3K and PKC, is involved in SH2-SH2-SH3 mediated NF-kappaB activation in these cells.