Initiation of protein synthesis in eukaryotes is important both mechanically, because it selects the translation reading frame, and biologically, because it is the primary site for regulation of translation. For approximately 95–98% of the cellular mRNAs, formation of an initiation complex follows a rather specific pathway that is broken down into seven discrete steps. Although almost 35 peptides in 12–14 translation initiation factors participate in this process, three appear to have dominant roles: eukaryotic initiation factor (eIF)3, in building a pool of 40S subunits; eIF2, in binding the initiator tRNA (tRNAi) to the 40S subunit; and eIF4F in activating the mRNA and binding it to the 40S subunit. This process requires both ATP and GTP. The resulting 80S initiation complex contains both the tRNAi and the mRNA, with the anticodon of the tRNAi correctly base paired with the initiating AUG code word. Regulation of translation focuses mostly on controlling the activity of either eIF2 or eIF4F and this regulation has different consequences. Reduction in eIF2 activity influences all mRNA approximately the same, whereas reduction in eIF4F activity drives competition between mRNAs.