To investigate the role of newly synthesized proteins during autophagic sequestration and degradation, the effects of protein synthesis inhibition on autophagic vacuole (AV) formation and degradation were analyzed. The inhibition of protein synthesis was found to separate autophagic sequestration from the delivery of lysosomal enzymes to (AVs). Pretreatment with cycloheximide for ≥ 3 h caused a drastic inhibition of autophagy-induced degradation. Surprisingly, morphological analyses showed that the inhibition of protein synthesis for up to 12 h did not block the formation of nascent AVs; however, it did prevent their conversion into degradative AVs. Using immunoperoxidase cytochemistry with an antibody against cathepsin D and labeling of lysosomes with endocytosed colloidal gold, we found that the nascent AVs that formed during prolonged cycloheximide pretreatment had not received lysosomal markers. The inhibition of autophagic degradation and lysosomal enzyme delivery were rapidly reversed following the removal of cycloheximide. These results suggest that there is a fairly rapid turnover of protein(s) that are necessary for lysosomal fusion, but that the initial formation of AVs is independent of new protein synthesis for a long period of time.