The microtubule inhibitor vinblastine causes accumulation of autophagic vacuoles in many cell types. In hepatocytes, many of the accumulated vacuoles are nascent, which has been interpreted to suggest that vinblastine acts by inhibiting the fusion of hydrolase-containing lysosomes with early autophagic vacuoles. However, our previous results suggested that, in Ehrlich ascites cells, vinblastine causes accumulation mainly of older autophagic vacuoles (AVs). This study was undertaken to further characterize the mode of action of vinblastine in these cells. The vinblastine-accumulated AVs were quantified by electron-microscopic morphometry. In addition, the effects of inhibitors of autophagic segregation (leucine, histidine, and 3-methyladenine) on the vinblastine-induced accumulation of autophagic vacuoles were studied. Protein degradation was measured using [14C]valine. Vinblastine caused accumulation of advanced autophagic vacuoles but did not increase the rate of protein degradation. The volume density of early vacuoles remained at the control level. The amino acids retarded but did not prevent the accumulation of autophagic vacuoles, whereas 3-methyladenine almost completely prevented the accumulation. The results suggest that in Ehrlich ascites cells vinblastine acts by inhibiting the maturation of advanced autophagic vacuoles into residual bodies and by stimulating the formation of new autophagic vacuoles. However, 3-methyladenine almost completely prevents the formation of new autophagic vacuoles in the presence of vinblastine. In conclusion, in Ehrlich ascites cells, vinblastine does not prevent the entry of hydrolases into autophagic vacuoles. This calls into question the importance of microtubules in the transport of lysosomal enzymes into autophagic vacuoles.