During inhibition of protein synthesis by the antibiotic enomycin at less than 5nm in the reticulocyte lysate system, polyribosomes disaggregated and the 80S ribosomes accumulated. At these concentrations little inhibition of chain elongation and release from the ribosomes was demonstrated. Enomycin caused an increase in the amount of 80S initiation complex as well as the 40S ribosomal subunit-Met-tRNA complex. The former complex could react with puromycin under the inhibiting conditions. Val-tRNA binding to the 80S ribosomes was not decreased by the antibiotic. However pactamycin-induced accumulation of the initial dipeptide (fMet-Val) was inhibited when the system was preincubated with enomycin and and fMet-tRnaf. Thus the preferential inhibition of the initial phase of protein synthesis by enomycin is made evident by its inhibition of the initial dipeptide synthesis.