3-Methyladenine is generally used as an inhibitor of autophagy [P. O. Seglen & P. B. Gordon (1982) Proc. Natl Acad. Sci. USA 79, 1889-1892]. Using isolated hepatocytes, we observed that 3-methyladenine has other effects as well. 1. 3-Methyladenine promoted glycogen breakdown and inhibited flux through phosphofructokinase and pyruvate kinase. These effects proved to be unrelated to inhibition of autophagic proteolysis and were caused by cAMP, which slightly increased in the presence of 3-methyladenine. 2. Addition of 3-methyladenine to intact hepatocytes increased the intralysosomal pH and caused a lower density of the lysosomal population upon centrifugation in a Percoll density gradient. No increase in the intralysosomal pH was effected by 3-methyladenine in isolated lysosomes.