The active protein components of initiation factor M2B (IF-M2B) have been resolved into two homogeneous factors. These proteins, IF-M2Balpha and IF-M2Bbeta, were purified 300- and 500-fold, respectively, with a yield of about 15% of the original starting activity. The low molecular weight (approximately 17,000) of these two proteins is in contrast with the much greater molecular weights that have been found for other initiation factors. IF-M2Balpha is also unique among the initiation factors in that it contains no tryptophan and is capable of self-association. Both proteins are required for model assays which utilize 40 S and 60 S subunits (poly(U)-directed polyphenylalanine synthesis or AUG-directed methionyl-puromycin synthesis). IF-M2Bbeta has been shown to be required for hemoglobin synthesis, however, the presence of high concentrations of IF-M2Balpha in the 100,000 X g lysate supernatant has precluded the demonstration of an IF-M2Balpha requirement in hemoglobin synthesis.