To kill mammalian cells, diphtheria toxin must be endocytosed and encounter a low pH within intracellular vesicles. The low pH initiates penetration of the catalytically active A fragment of the toxin through a membrane and into the cytosol where the A fragment arrests protein synthesis. To investigate whether penetration occurred through a prelysosomal or a lysosomal membrane, we studied the effect of low temperature on the entry of the toxin into the cytosolic and lysosomal compartments. The toxin arrested protein synthesis at 15 degrees C, indicating entry into the cytosol; however, access to lysosomes was apparently blocked at 15 degrees C, suggesting that the toxin had encountered a low pH before reaching lysosomes and had penetrated a prelysosomal membrane. To further investigate the possibility of prelysosomal acidification, we measured the time required for the toxin to encounter a low pH after endocytosis. Acidification occurred within 3 to 4 min after the toxin was internalized into vesicles. This interval is consistent with prelysosomal acidification since the entry of endocytosed ligands into secondary lysosomes usually takes more than 3 to 4 min.