During protein synthesis, tRNAs successively occupy three sites on the ribosome: A (aminoacyl), P (peptidyl), and E (exit). A proposed model based on observations of stalled ribosomes describes two distinct states before mRNA and tRNA translocation (PRE-translocation): a classical state in which the tRNAs occupy the same sites in the large and small ribosomal subunits (A/A and P/P), and a hybrid state in which the tRNAs occupy different sites in each subunit (A/P and P/E). To determine whether the exchange and partition between the classical and hybrid PRE states of stalled ribosomes is important for actively translating ribosomes, we conducted single molecule FRET experiments using ribosomes containing Cy5-labeled large subunit protein L11, and Val-tRNAVal(Cy3) or Phe-tRNAPhe(Cy3) in the A-site. We monitored FRET in stalled PRE complexes in the absence of EF-G•GTP, and during ongoing translation in the presence of EF-G•GTP. The mRNA contained a variable number (0, 4, or 8) of Tyr codons preceding Phe and Val codons. In stalled ribosomes, the histogram of L11-tRNA FRET values showed two peaks corresponding to the classical and hybrid states. In contrast, in actively translating ribosomes, the FRET histogram showed only one major peak, with a FRET efficiency that was intermediate between the two values for the stalled complexes. This distribution could correspond to an intermediate structure unique to active turnover, or to a transient state that, in the presence of EF-G, undergoes translocation more rapidly than it converts into the canonical classical and hybrid states. These results suggest that the PRE-translocation ribosomal state during active translation is distinct from the classical and hybrid states observed in stalled ribosomes, consistent with earlier results reported by our group (Rosenblum et al., JACS 2013). Supported by NIH grant GM080376 and AHA fellowship 12POST8910014 to CC.