The activities of several lysosomal enzymes have been determined in erythrocytes from human donors with and without spleens. The activities of the lysosomal enzymes β-N-acetylglucosaminidase (EC 22.214.171.124) and β-galactosidase (EC 126.96.36.199) are strikingly increased in erythrocytes from splenectomized donors, especially those with high reticulocyte counts. In contrast, erythrocytes from normal subjects have low levels of these enzymes regardless of the reticulocyte count. The activity of glucose-6-phosphate dehydrogenase (EC 188.8.131.52), a soluble enzyme, was the same in erythrocytes from these two groups of subjects as was the total activity of acid β- glycerophosphate phosphohydrolase (EC 184.108.40.206), a characteristic lysosomal enzyme. This discrepancy was resolved by gel filtration studies which showed that erythrocytes from normal subjects contain two acid phosphatases while erythrocytes from splenectomized patients contain three different acid phosphatases. The third acid phosphatases is localized to membrane-bounded particles. Each of the three acid phosphotases has a different molecular weight and responds differently to various substrates and inhibitors, Mg2+, pH, and temperature. These studies provide biochemical evidence for the existence of lysosomes in ery erythrocytes and demonstrate that the spleen determines the level of lysosomes in these cells.