Secretion of lysosomal enzymes into the extracellular surroundings has been observed in many eukaryotic cells. We studied the activity of lysosomal enzymes in different subcellular fractions of Tetrahymena thermophila to get more insight into this general phenomenon. By density gradient centrifugation a light and a dense fraction of lysosomal particles were found. Electron microscopy revealed that the light fraction mainly consists of cell surface membranes. By immunostaining a lysosomal enzyme (beta-hexosaminidase) was detected on the plasma membrane. The Triton X-114 assay showed that the light fraction as well as purified cilia (an enriched source of plasma membrane) contain lysosomal enzymes predominantly covalently bound to the membrane. The dense fraction contains both membrane-bound and soluble forms of lysosomal enzymes. By labeling phagosomes/phagolysosomes with magnetic particles the dense fraction can be subdivided into two lysosomal vesicle populations: phagolysosomes and a further population of lysosomal vesicles which can not be labeled. The relationship between membrane-bound and soluble enzyme forms in phagolysosomes and this unlabeled vesicle population is different: In phagolysosomes 80% of the acid phosphatase and 20% of the beta-hexosaminidase are membrane-bound, whereas in the unlabeled vesicles 42% of the acid phosphatase and 8% of the beta-hexosaminidase are bound to the membrane. Furthermore, we present results suggesting that the unlabeled vesicle population of the dense fraction is the source of secreted lysosomal enzymes. A working model summarizing our present knowledge about the connection of the three pools of lysosomal enzymes in Tetrahymena is presented.