Ethanol (0.21-0.84 M) added to rabbit reticulocyte lysates results in a 42-89% decrease of polypeptide synthesis following a 60-min incubation at 30 degrees C. The onset of inhibition is preceded by a 5-15-min lag. Eukaryotic initiation factor 3 (eIF-2) (85% pure, 0.42-4.2 micrograms added/30-microliters assay) or high concentrations of cAMP, MgGTP, and glucose 6-phosphate (0.55-5.0 mM) partially reverse the inhibition was observed with fructose 6-phosphate (0.55 mM). Ethanol was also shown to directly inhibit ternary complex formation between eIF-2, GTP, and initiator Met-tRNA (50% inhibition with 0.5 M ethanol). The inhibitory effect of ethanol on polypeptide synthesis, however, appears to be independent of its effect on ternary complex formation, and may be related to the activation of a translational inhibitor. This tentative conclusions is based on the following results. First, when the postribosomal supernatant or unfractionated lysate is incubated with ethanol, the supernatant or lysate becomes inhibitory to polypeptide synthesis following a 60-min incubation with ethanol at 30 degrees C. Second, when ethanol-treated postribosomal supernatant is chromatographed on DEAE-cellulose, an inhibitory activity is observed. The peak of inhibition coincides with the elution position of a translational inhibitor termed heme-controlled repressor. Third, pro-heme-controlled repressor becomes more inhibitory to protein synthesis when incubated with ethanol. These findings suggest that ethanol may activate a translational inhibitor by affecting the proper conformational change of a dormant inhibitor.