Eukaryotic initiation factor 3 (eIF3) is a large multisubunit complex that stabilizes the ternary complex, eIF2 x GTP x tRNA(Met)i and promotes mRNA binding to the 40 S ribosomal subunit. eIF3 also functions as a ribosome subunit anti-association factor. The molecular mechanisms by which eIF3 exerts these functions are poorly understood. We describe here the cloning of the cDNA encoding the human homologue of the yeast eIF3 subunit Prt1. The human PRT1 cDNA encodes a protein of predicted molecular mass of 98.9 kDa that migrates at 116 kDa on SDS-polyacrylamide gels. Human and yeast Prt1 share 31% identity and 50% similarity at the amino acid level. The homology is distributed throughout the entire protein, except for the amino terminus, and is particularly high in the central portion of the protein, which contains a putative RNA recognition motif. hPrt1 is recognized by an antibody raised against eIF3, and an affinity-purified antibody to recombinant hPrt1 recognizes a protein migrating at 116 kDa in a purified eIF3 preparation. Far Western analysis shows that hPrt1 interacts directly with the p170 subunit of eIF3. Mapping studies identify the RNA recognition motif as the region required for association with p170. Taken together, these experiments demonstrate that hPrt1 is a component of eIF3. Our data, combined with those of Hershey and co-workers, suggest that mammalian eIF3 is composed of at least 10 subunits: p170, p116 (hPrt1), p110, p66, p48, p47, p44, p40, p36, and p35.