Knowledge of the cellular distribution and subcellular localization of mannose-terminal glucocerebrosidase after intravenous infusion is necessary for understanding the efficacy of targeted enzyme replacement therapy for Gaucher's disease. Selective uptake of mannose-terminal glucocerebrosidase by Kupffer cells in rat liver has been previously demonstrated biochemically. In this study we used immunohistochemical and immunogold labeling techniques to provide direct visual proof for the localization of the delivered enzyme. Light microscopy confirmed biochemical data identifying non-parenchymal cells as the primary target of the modified glucocerebrosidase. Using a primary antibody specific for glucocerebrosidase and a secondary gold-conjugated antibody, we used immunoelectron microscopy to quantify the extent and distribution of exogenous enzyme in various cell types in rat liver and its localization within their respective subcellular organelles. Thirty minutes after intravenous administration of mannose-terminal glucocerebrosidase, enzyme was localized primarily in lysosomes of Kupffer cells. Of eight intact Kupffer cells counted, 16 of 21 lysosomes (78%) contained immunogold conjugates (average concentration 293 gold particles/micron 2). Of 589 particles counted in these lysosomes, 485 (82%) were localized within the lumen of the lysosome; only 104 (18%) were membrane-associated. Five of the 21 lysosomes counted were negative for gold. No gold particles were found in the mitochondria of Kupffer cells and very few particles (8.2/microns 2) were found over the nucleus. The density of gold particles was also low over the nucleus (7.2/microns 2), mitochondria (8.8/microns 2), and lysosomes (7.9/microns 2) of hepatocytes. No specific labeling was observed in erythrocytes, platelets, lymphocytes, pit cells, fat-storing cells, or bile duct. Background labeling of control liver sections from rats receiving saline injection was 8.2 +/- 1.4 gold particles/microns 2. We conclude that mannose-terminal glucocerebrosidase is delivered to the lysosomes of Kupffer cells in liver and that it is distributed both within the lumen (82%) and over the membrane (18%) of the lysosome, with a slight preferential association with the membrane. These findings may provide insights into the design of more effective therapeutic enzyme preparations for the treatment of Gaucher's disease.