Lysosomes are responsible for the degradation of macromolecules derived from the cell exterior by endecytosis, or from within the cell by autophagy. While our knowledge of the biosynthesis and targeting of lysosomal hydrolases is considerable, much less is known about the lysosomal membrane itself. To identify the lysosomal membrane proteins that mediate these functions, we have isolated lysosomes from amebae and injected them into mice to produce monoclonal antibodies (MAbs). We produced nine MAbs against Dictyostelium lysosomes from the batches of fused cells. Among them, three MAbs were specific to lysosomal membrane and gave a strong signal, and thus used in this study. The MAbs specifically reacted with a single protein band of 27 kd and stained a lysosome-like structure by immunofluorescence microscopy. To identify the antigen that the MAbs recognize, we processed differential centrifugation with whole-cell extract of Dictyostelium and traced p27 protein by activity assay of organelle marker enzyme. We showed that p27 is one component of the lysosomal system on the basis of comigration with a lysosomal marker enzyme. We also demonstrated that the 27-kd lysosomal protein is a tightly bound integral membrane protein by using a phase separation method of Triton X-114.