The Raf/MEK/ERK (extracellular regulated kinase) signal transduction pathway controls the ability of cells to respond to proliferative, apoptotic, migratory and differentiation signals. We have investigated the combined contribution of A-Raf and Raf-1 isotypes to signalling through this pathway by generating mice with knockout mutations of both A-raf and raf-1 genes. Double knockout (DKO) mice have a more severe phenotype than single null mutations of either gene, dying in embryogenesis at E10.5. The DKO embryos show no changes in apoptosis, but staining for Ki67 indicates a generalized reduction in proliferation. DKO mouse embryonic fibroblasts (MEFs) exhibit a delayed ability to enter S phase of the cell cycle. This is associated with a reduction in levels of transiently induced MEK and ERK phosphorylation and reduced expression of c-Fos and cyclin Dl. Levels of sustained ERK phosphorylation are not significantly altered. Thus, Raf-1 and A-Raf have a combined role in controlling physiological transient ERK activation and in maintenance of cell cycle progression at its usual rate.Oncogene advance online publication, 25 April 2005; doi:10.1038/sj.onc.1208707