Reviews (1, 2) of studies on lysosomes suggest that these organelles may play an important role in normal processes and in diverse pathologic conditions. Since lysosomes of different tissues may vary to a considerable extent, it is important to identify and characterize these organelles in the particular tissue to be investigated.The presently available evidence for the presence of lysosomes in the epidermis is morphologic or histochemical. In electron micrographs, subcellular structures which resemble lysosomes have been seen in keratinocytes (3), in Langerhans cells (4, 5), and in intraepider-mal eccrine duct cells (6). Subcellular particles containing acid phosphatase have also been demonstrated in electron micrographs of keratinocytes of normal (7) and abnormal epidermis (8).Diengdoh (9) reported latency (time-related appearance) of acid phosphatase in mouse epidermis by using a histochemical technique and the light microscope. However, Eisen and coworkers (10) were unable to confirm this in human epidermis.Morphologic and histochemical methods cannot fulfill all the criteria for lysosomes (1). We have used biochemical methods for the demonstration of lysosomes in the epidermis of man and of the hairless mouse.METHODSHuman skin was obtained from the abdomen at autopsy or from surgical amputations. Skin from the backs of hairless mice was also used. Epidermis was removed by the stretch method (11) and was immediately chilled in ice-cold, 0.3M sucrose buffered with 0.01M phosphate buffer, pH 7.3. The tissue was homogenized in small batches for 10-second intervals at one-third to one-half speed in a VirTis Model 23 with the micro attachment;the homogenate was filtered through one or two layers of cheesecloth to remove gross tissue fragments. The cloudy suspension was separated into three fractions, designated “nuclear,” “lysosomal/ and “supernatant” fractions, by the procedure of differential centrifugation indicated in Table I.Acid phosphatase was determined fluorometri-cally by adapting the procedure of Campbell and Moss (12) to the Turner Model 111 fluorometer with primary filter no. 7-60 (360 m/*) and secondary filter no. 47B (415 m/0. Acid phosphatase activity was used to determine latency and specific activity. /3-Glucuronidasev was assayed by a modification of the method of Fishman and coworkers (13), aryl sulfatase was determined by the method of Roy (14), and cathepsin was determined by the method of Gianetto and de Duve (15); these enzymes were measured only on ho-mogenates because of the relative insensitivity of the assay procedures for their detection.Two technics were used to study the latent properties of the isolated lysosomes. According to the method of de Duve and co-workers (16), the lysosome suspension was incubated with substrate with or without exposure to disrupting conditions (for example, Triton-X-100). Thus, the access of substrate to enzymes still associated with granules is measured. This assay necessitates short incubation times since the pH of assay is sufficient to cause disruption of lysosomes. The acid phosphatase assay was the only procedure sensitive enough for this technic. By following the alternate method of Dingle (17), the particles were removed by centrifugation after pre-incubation with disrupting agents. The enzyme activity found in the supernate measures the release of enzyme from the granules caused by the test conditions. Protein was determined in the fractions by the method of Lowry and co-workers (18). Enzyme activities of the three fractions were measured after treatment with 0.1% Triton-X-100 for calculat 1SUMMARY Four biochemical tests have demonstrated the presence of lysosomes in the epidermis of man and of the hairless mouse: (1) presence of acid hydrolases, (2) sedimentability, (3) increased specific activity, (4) structure-linked latency. Epidermal homogenates from man and from the hairless mouse have been shown to contain four lysosomal enzymes: acid phosphatase, cathepsin, /3-glucuronidase, and aryl sulfatase. Acid phosphatase activity was used to demonstrate sedimentability of the lysosomal fraction and structure-linked latency. Acid pH, hypotonic solution, and freezing and thawing did not solubilize epidermal lysosomes. Triton- X-100 produced time-dependent solubiization (latency) of epidermal lysosomes by two methods. . De Duve, Christian: The lysosome concept.