mRNA analogues containing several 4-thiouridine (thio- U) residues at selected positions were prepared by T7-transcription. The spacer region between the Shine-Dalgarno sequence and the AUG codon consisted of four or eight bases with a single thio-U at a variable position; alternatively, cro-mRNA analogues were used carrying the thio-U substituted spacer sequence UUGU. The mRNAs were bound to E.coli ribosomes, and-after irradiation-the sites of crosslinking to 16S RNA were analysed. Three cross-links to the 16S RNA from the spacer region were observed, namely to positions 665, 1360, and a site close to nucleotide 1530. The cross-links were formed in different amounts in the presence or absence of tRNAtMet, and were observed from thio-U residues located at various positions within the spacer sequence, although in the presence of tRNA they were in general stronger from positions close to the Shine - Dalgarno end of the spacer. The cross-linking behaviour in this upstream area of the mRNA is thus rather different in character from the previously published pattern in the downstream area. From considerations of structural conservation in small subunit RNA, we propose that both the upstream and downstream cross-links to 16S RNA reflect a universal mRNA path through the ribosome.