Seven amino acid peptides were tested as in vitro inhibitors of oncogenic Ras-Raf association. The sequences of these peptides were derived from the H-Ras effector region (amino acids 25 to 51) and the Ras binding domain of Raf-1 (amino acids 64 to 105). Eleven out of the twenty-one Ras 7-mers tested inhibited formation of the Ras-Raf complex by at least 20% at 100 microM. The most potent of these inhibitory peptides contained the effector residues 32 to 37 or 40 to 45. Of the Raf-1 peptides tested, only the 94-ECCAVFR-100 and 95-CCAVFRL-101 peptides were significant inhibitors of Ras-Raf binding. The 95-101 Raf peptide had an IC50 value of 7 microM and also inhibited Ras-RalGDS binding. Analysis of the 95-101 peptide showed that its inhibitory activity required at least one cysteine followed by several hydrophobic residues. Our results demonstrate the feasibility of using small molecules as inhibitors of Ras protein-protein interactions.