A critical event in a signal transduction pathway downstream of receptor tyrosine kinases is the physical association of GTP-liganded Ras with the serine/threonine kinase, Raf-1. The binding of Raf-1 to Ras results in translocation of the kinase to the plasma membrane and facilitates its activation by an unknown mechanism. A deletion mutagenesis approach was employed to elucidate critical sequences in Raf-1 necessary for binding to Ras and to resolve seemingly contradictory data in the literature. While an N-terminal fragment consisting of residues 2-130 of Raf-1 was able to bind Ras, residues 131-147 were found to be critically important for conferring high affinity binding to Ras. Surprisingly, a second domain between residues 52-64 was an essential element for Raf-Ras interaction, although it did not appear to form an independent binding site for Ras. These findings may prove useful for the design of peptides or peptidomimetic drugs for the modulation of Raf-Ras interaction in neoplastic disorders.