The effect of wortmannin on the trafficking of the mannose 6- phosphate/insulin-like growth factor II receptor (MAn-6-P/IGF-II receptor) and its ligand β-glucuronidase has been determined in murine L cells and normal rat kidney cells. The drug induced a 90% decrease in the steady-state level of the Man-6-P/IGF-II receptor at the plasma membrane without affecting the rate of internalization, indicating that the return of receptor from endosomes to the plasma membrane is retarded. Wortmannin also slowed the movement of receptor from endosomes to the trans-Golgi network by about 60%. Such a kinetic block would dramatically reduce the number of Man-6-P/IGF-II receptors in the trans-Golgi network, which could account for the previously described hypersecretion of procathepsin D induced by wortmannin. In addition, the drug slowed delivery of endocytosed β-glucuronidase from endosomes to dense lysosomes. These data, taken together with the published reports of others, indicate that wortmannin inhibits membrane trafficking out of multiple compartments of the endosomal system and suggest a role for phosphatidylinositol 3-kinase in regulating these processes.