3-Methyladenine is a specific inhibitor of autophagic/lysosomal protein degradation in isolated rat hepatocytes. It inhibits endogenous protein degradation by about 60% at 5 mM, without affecting exogenous protein degradation, protein synthesis, or intracellular ATP levels. It acts specifically on the autophagic/lysosomal pathway, as it is not affected by amino acids or a lysosomotropic amine (propylamine). The inhibition of protein degradation is accompanied by a significant depression of intracellular amino acid levels, indicating that the effect is not mediated by amino acids. The ability of 3-methyladenine to suppress the formation of autophagosomes suggests it is a specific inhibitor of autophagy. Autophagy and lysosomes are involved in both exogenous and endogenous protein degradation. Amino acids suppress the first step in the autophagic/lysosomal pathway—autophagosome formation. 3-Methyladenine, like amino acids, inhibits autophagosome formation independently of amino acids. It was found that 3-methyladenine inhibits endogenous protein degradation more effectively than other purines, with a high selectivity for degradation. 3-Methyladenine does not affect the degradation of exogenous protein (asialofetuin), indicating its effect is on a prelysosomal step unique to autophagy. It does not inhibit protein synthesis or intracellular ATP levels, even at high concentrations. This suggests that the effect of 3-methyladenine is not mediated by changes in amino acid availability or energy metabolism. The results indicate that 3-methyladenine is a specific inhibitor of autophagy in hepatocytes. It may be useful in studying the role of autophagy under various physiological conditions and in understanding the biochemistry of autophagy. The ability to specifically suppress autophagy may have practical applications in maintaining and growing cultured cells or in vivo under conditions where autophagy contributes to excessive tissue wasting.3-Methyladenine is a specific inhibitor of autophagic/lysosomal protein degradation in isolated rat hepatocytes. It inhibits endogenous protein degradation by about 60% at 5 mM, without affecting exogenous protein degradation, protein synthesis, or intracellular ATP levels. It acts specifically on the autophagic/lysosomal pathway, as it is not affected by amino acids or a lysosomotropic amine (propylamine). The inhibition of protein degradation is accompanied by a significant depression of intracellular amino acid levels, indicating that the effect is not mediated by amino acids. The ability of 3-methyladenine to suppress the formation of autophagosomes suggests it is a specific inhibitor of autophagy. Autophagy and lysosomes are involved in both exogenous and endogenous protein degradation. Amino acids suppress the first step in the autophagic/lysosomal pathway—autophagosome formation. 3-Methyladenine, like amino acids, inhibits autophagosome formation independently of amino acids. It was found that 3-methyladenine inhibits endogenous protein degradation more effectively than other purines, with a high selectivity for degradation. 3-Methyladenine does not affect the degradation of exogenous protein (asialofetuin), indicating its effect is on a prelysosomal step unique to autophagy. It does not inhibit protein synthesis or intracellular ATP levels, even at high concentrations. This suggests that the effect of 3-methyladenine is not mediated by changes in amino acid availability or energy metabolism. The results indicate that 3-methyladenine is a specific inhibitor of autophagy in hepatocytes. It may be useful in studying the role of autophagy under various physiological conditions and in understanding the biochemistry of autophagy. The ability to specifically suppress autophagy may have practical applications in maintaining and growing cultured cells or in vivo under conditions where autophagy contributes to excessive tissue wasting.