The effect of 3-methyladenine on transport from endosomes to lysosomes was studied in rat embryonic and mouse 3T3 fibroblasts. Subcellular fractionation in 27% Percoll gradients showed that the pre-endocytosed (5 min pulse) horseradish peroxidase (HRP) was not transported from endosomes to dense lysosomes in cells chased in the presence of 10 mM 3-methyladenine. However, fractionation in 20% Percoll gradients, which separated early endosomes from late endosomes and lysosomes, as well as light and electron microscopic experiments, showed that HRP was transported from early endosomes to the perinuclear late endosomes. Immunoprecipitation of metabolically labeled cells was used to study the biosynthetic processing of a lysosomal proteinase, cathepsin L. The results showed that the early processing of the precursor to the intermediate form was not affected by 3-methyladenine, while the late processing of the intermediate to the mature form was retarded. In addition, immunofluorescence labeling showed that 3-methyladenine treatment caused accumulation of cathepsin L in the perinuclear area. Another lysosomal enzyme, beta-glucuronidase, was normally distributed in both perinuclear and peripheral vesicles which indicated that the localization of lysosomes was not altered. The results thus suggest that the late processing of cathepsin L was inhibited because transport from perinuclear endosomes to lysosomes was retarded. In conclusion, both endocytic pulse-chase experiments and immunoprecipitation of metabolically labeled cathepsin L indicate that 3-methyladenine inhibits transport from late endosomes to mature lysosomes in both rat and mouse fibroblasts.