Eukaryotic translation initiation factor 3 (eIF3), which plays an essential role in initiation of protein synthesis, was purified from rabbit reticulocyte lysates using an assay that specifically measures its ability to stimulate the binding of Met-tRNAf (as a Met-tRNAf.eIF2.GTP ternary complex) to 40 S ribosomal subunits. Purified eIF3 consisted of six major polypeptides of molecular masses 110, 67, 42, 40, 36, and 35 kDa but lacked the 170-kDa polypeptide reported to be a constituent of other eIF3 preparations. Characterization of purified eIF3 lacking the 170-kDa polypeptide showed that the eIF3-mediated 40 S initiation complex formed in the presence of AUG codon efficiently joined 60 S ribosomal subunits in an eIF5-dependent reaction to form a functional 80 S initiation complex. eIF3, which was originally bound to the 40 S initiation complex, was released from the 40 S subunit during the subunit joining reaction. Additionally, chicken antibodies raised against rabbit reticulocyte eIF3 were used to immunochemically characterize eIF3 subunits and to isolate a 3.1-kilobase pair human cDNA that encodes the p110 subunit of mammalian eIF3. The derived amino acid sequence (calculated Mr 95,214) shows that the p110 subunit is the mammalian homologue of Saccharomyces cerevisiae protein Prt1p, a subunit of yeast eIF3.