Mitogenic signaling involves protein kinases that phosphorylate the mitogen-activated protein kinase (MAPK) activator, MEK. In rats, basal hepatic MEK kinase activity is low in vivo in both adult rats and late gestation fetal rats, and is markedly stimulated by intraperitoneal administration of epidermal growth factor (EGF). The level of stimulated MEK phosphorylating activity is approximately 15 times higher in fetal liver than in adult liver. To identify regulated forms of the two categories of MEK kinase, Raf and MEKK, Western immunoblotting, immunoprecipitation kinase assays and immunodepletion studies were performed. Western immunoblotting confirmed that Raf-1, A-Raf, B-Raf, MEKK1 and MEKK2 were present at similar levels in E19 and adult liver. However, specific immunoprecipitation kinase assays did not detect any kinases that could account for marked EGF sensitivity or the higher level of activity in E19 fetuses. Immunodepletion studies produced a marked reduction in immunoreactive Raf/MEKK content and activity, but a minimal decrease in the ability of chromatography fractions to phosphorylate and activate recombinant MEK-1. Our results indicate that hepatic, EGF-sensitive MEK kinase activity may reside with a previously unidentified and physiologically relevant form of Raf and/or MEKK.