Severe muscle wasting is a characteristic feature of sepsis. We have previously established that the rate of protein synthesis in muscles composed of fast-twitch fibers is severely diminished in response to sepsis. The present studies investigate the biochemical reactions responsible for the decreased rate of protein synthesis using gastrocnemius from control and septic rats perfused in situ. Analysis of free ribosomal subunits indicated peptide-chain initiation was impaired by infection. To characterize biochemical reactions in the pathway of peptide-chain initiation affected, the effect of sepsis on the incorporation of initiator [35S]methionyl-tRNA (met-tRNA(imet)) into the 40S initiation complex was examined. Sepsis caused a 65% decrease in the binding of radiolabelled met-tRNA(imet) to the 40S initiation complex compared with controls. The binding of met-tRNA(met) to the 40S ribosome is regulated by eukaryotic initiation factor eIF-2B, whose activity can be modulated in part by the redox state of pyridine dinucleotides. The mean cytoplasmic NADH/NAD+ ratio was increased 2 fold in sepsis, while the NADPH/NADP+ ratio was unchanged. These findings identify the formation of the 40S initiation complex as a defect in the protein synthesis machinery during sepsis. The decreased formation of the 40S initiation complex in muscle could not be explained by changes in the cytoplasmic redox state.