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With the recent availability of high-resolution structures of bacterial ribosomes, studies of ribosome-catalyzed protein biosynthesis are now focusing on the nature of conformational changes that occur as the ribosome exerts its complex catalytic function. Photocrosslinking can be relevant for this purpose by providing clues to ribosomal structural fluctuations and dynamics. Here we describe crosslinking experiments on 70S ribosomes using two photolabile anticodon stem-loop derivatives of Escherichia coli tRNAPhe carrying a 4-thiouridine in either position 33 or 37 and denoted Ph-ASLs. One or both of these Ph-ASLs bind to the tRNA A-, P-, and E-sites on the ribosome, with both binding to and photocrosslinking from the E-site showing strong dependence on the presence of a tRNA in the P-site. Both Ph-ASLs crosslink to the extreme 3'-end of 16S rRNA from both the P- and E-sites, providing direct confirmatory evidence in solution for the folding back of the 3'-end toward the decoding region. This suggests that the 3'-end of 16S rRNA may act as a switch in controlling mRNA access to the decoding center, a phenomenon of potential relevance for the translation of leaderless mRNA. E-site bound Ph-ASLs also form photocrosslinks to nucleotides 1395-1398, 1399-1400, and 1491-1494 at the top of helix 44 of 16S rRNA, indicating movement of the decoding center from a position between the A- and P-sites seen in the crystal structure to one neighboring the E-site.