Ribosomal protein S1 is known to play an important role in translational initiation, being directly Involved In recognition and binding of mRNAs by 30S ribosomal particles. Using a specially developed procedure based on efficient crossllnking of S1 to mRNA induced by UV irradiation, we have identified S1 binding sites on several phage RNAs in preinltiation complexes. Targets for S1 on Qβ and fr RNAs are localized upstream from the coat protein gene and contain oligo(U)-sequences. In the case of Qβ RNA, this S1 binding site overlaps the S-site for Qβ replicase and the site for S1 binding within a binary complex. It is reasonable that similar U-rich sequences represent S1 binding sites on bacterial mRNAs. To test this idea we have used E. coli ssb mRNA prepared in vitro with the T7 promoter/RNA polymerase system. By the methods of toeprinting, enzymatic footprinting, and UV crosslinking we have shown that binding of the ssb mRNA to 30S ribosomes is S1-dependent. The oligo{U)-sequence preceding the SD domain was found to be the target for S1. We propose that S1 binding sites, represented by pyrimidine-rich sequences upstream from the SD region, serve as determinants involved In recognition of mRNA by the ribosome. © 1991 Oxford University Press.