Eukaryotic initiation factor (eIF) 1 maintains the fidelity of initiation codon selection and enables mammalian 43S preinitiation complexes to discriminate against AUG codons with a context that deviates from the optimum sequence GCC(A/G)CCAUGG, in which the purines at (-)3 and (+)4 positions are most important. We hypothesize that eIF1 acts by antagonizing conformational changes that occur in ribosomal complexes upon codon-anticodon base-pairing during 48S initiation complex formation, and that the role of (-)3 and (+)4 context nucleotides is to stabilize these changes by interacting with components of this complex. Here we report that U and G at (+)4 both UV-cross-linked to ribosomal protein (rp) S15 in 48S complexes. However, whereas U cross-linked strongly to C(1696) and less well to AA(1818-1819) in helix 44 of 18S rRNA, G cross-linked exclusively to AA(1818-1819). U at (-)3 cross-linked to rpS5 and eIF2alpha, whereas G cross-linked only to eIF2alpha. Results of UV cross-linking experiments and of assays of 48S complex formation done using alpha-subunit-deficient eIF2 indicate that eIF2alpha's interaction with the (-)3 purine is responsible for recognition of the (-)3 context position by 43S complexes and suggest that the (+)4 purine/AA(1818-1819) interaction might be responsible for recognizing the (+)4 position.