In archaeal translation initiation, a preinitiation complex (PIC) made up of aIF1, aIF1A, the ternary complex (TC, e/aIF2-GTP-Met-tRNAiMet) and mRNA bound to the small ribosomal subunit is responsible for start codon selection. Many archaeal mRNAs contain a Shine-Dalgarno (SD) sequence allowing the PIC to be prepositioned in the vicinity of the start codon. Nevertheless, cryo-EM studies have suggested local scanning to definitely establish base pairing of the start codon with the tRNA anticodon. Here, using fluorescence anisotropy, we show that aIF1 and mRNA have synergistic binding to the Pyrococcus abyssi 30S. Stability of 30S:mRNA:aIF1 strongly depends on the SD sequence. Further, toeprinting experiments show that aIF1-containing PICs display a dynamic conformation with the tRNA not firmly accommodated in the P site. AIF1-induced destabilization of the PIC is favorable for proofreading erroneous initiation complexes. After aIF1 departure, the stability of the PIC increases reflecting initiator tRNA fully base-paired to the start codon. Altogether, our data support the idea that some of the main events governing start codon selection in eukaryotes and archaea occur within a common structural and functional core. However, idiosyncratic features in loop 1 sequence involved in 30S:mRNA binding suggest adjustments of e/aIF1 functioning in the two domains.