Fibroblast growth factors (FGFs) play a critical role in pituitary development and in pituitary tumor formation and progression. We have previously characterized FGF signal transduction and regulation of the tissue specific rat PRL (rPRL) promoter in GH4 pituitary cells. FGF induction of rPRL transcription is independent of Ras, but mediated by a PKC-dependent activation of MAP kinase (ERK). Here we demonstrate a functional role for the Rho family monomeric G-protein, Rac1, in FGF regulation of PRL gene expression via an atypical signaling pathway. Expression of dominant negative Rac, but not RhoA or Cdc42, selectively inhibited FGF-induced rPRL promoter activity. Moreover, expression of dominant negative Rac also attenuated FGF-2 and FGF-4 stimulation of MAPK (ERK). However, in contrast to other Rac-dependent signaling pathways, FGF activation of rPRL promoter activity was independent of the JNK and phosphoinositide 3-kinase/Akt cascades. FGFs failed to activate JNK1 or JNK2 and expression of dominant negative JNK or Akt constructs did not block FGF-induced PRL transcription. Consistent with the role of PKC in FGF regulation of PRL gene expression, activation of the rPRL promoter was blocked by an inhibitor of phospholipase C activity. FGF treatment also induced rapid tyrosine phosphorylation of PLC in a Rac-dependent manner. These results suggest that FGF-2 and FGF-4 activate PRL gene expression via a novel Rac1, PLC, PKC and ERK cascade, independent of PI3 Kinase and JNK.