Abstract: Eukaryotic initiation factor 3 (eIF3) is an essential, highly conserved multi-protein complex that is a key component in the recruitment and assembly of the translation initiation machinery. To better understand the molecular function of eIF3, we examined its composition and phosphorylation status in Saccharomyces cerevisiae. The yeast eIF3 complex contains five core components: Rpg1, Nip1, Prt1, Tif34, and Tif35. 2-D LC/MS/MS mass spectrometry analysis of affinity purified eIF3 complexes showed that several other initiation factors (Fun12, Tif5, Sui3, Sui2, Gcd11, Pab1, Hcr1, and Sui1) and the casein kinase 2 complex (CK2) co-purify. In vivo metabolic labeling of proteins with 32P revealed that Nip1 and Tif5 are phosphorylated. Using 2-D LC/MS/MS analysis of eIF3 complexes, we identified Prt1 phosphopeptides indicating phosphorylation at S61 and T746 and a Tif5 phosphopeptide with phosphorylation at T191. Additionally, we used immobilized metal affinity chromatography (IMAC) to enrich for eIF3 phosphopeptides and tandem mass spectrometry to identify phosphorylated residues. We found that three CK2 consensus sequences in Nip1 are phosphorylated: S98, S99, and S103. Using in vitro kinase assays, we showed that CK2 phophorylates Nip1 and that a synthetic Nip1 peptide containing S98, S99, and S103 competitively inhibits the reaction. Replacement of these three Nip1 serines with alanines causes a slow-growth phenotype.