The ribosome must accurately translocate mRNA to maintain the reading frame. Here, we monitor the position of mRNA within the ribosome before and after EF-G-catalyzed translocation near the initiation site. When a deacylated tRNA that is translocated to the 30S P site recognizes other nearby codons, movement of tRNA and mRNA often becomes uncoupled. Instead of moving in the 5 direction by 3 nucleotides, the mRNA slips backward, repositioning the tRNA on an out-of-frame codon more optimally spaced from the Shine-Dalgarno sequence. In contrast, when peptidyl-tRNA or its analog (N-acetyl-aminoacyl-tRNA) is translocated in the same context, translocation of mRNA is highly accurate. If aminoacyl-tRNA is translocated, an intermediate level of translocational accuracy is observed. Thus, translocational accuracy depends on the acylation state of the tRNA entering the 30S P site.