Eukaryotic initiation factor-3 (eIF-3) plays a pivotal role in the initiation phase of protein synthesis where it promotes dissociation of 80 S ribosomes into subunits, stabilizes methionyl-tRNAi binding to 40 S ribosomal subunits, and is required for mRNA binding. Mammalian eIF-3 is comprised of eight subunits, but no mammalian cDNA encoding these proteins has been cloned and sequenced, nor has the corresponding factor been characterized in yeast. Since many initiation factors are strongly conserved between mammalian and yeast systems, we employed a mammalian assay for initiation, the synthesis of methionyl-puromycin, to detect eIF-3 activity in yeast subcellular fractions. Yeast eIF-3 was purified from the high salt wash of ribosomes by Superose 6 molecular sieve and MonoS ion exchange chromatography. Yeast eIF-3 contains eight subunits with masses of 16, 21, 29, 33, 39, 62, 90, and 135 kilodaltons all of which coelute with an apparent mass of 550 kilodaltons from the Superose 6 column. Immunoblotting shows that the 90-kDa subunit corresponds to the product of the PRT1 gene whose mutant form, prt1-1, exhibits destabilization of methionyl-tRNAi binding to 40 S ribosomal subunits. eIF-3, and specifically the 62-kDa subunit, bind to RNA. These biochemical approaches to defining yeast eIF-3 complement genetic methods so far used in characterizing the initiation factors and provide another route to defining the yeast translational machinery.