The intracellular pathway for secretion of thymulin, a thymic hormone, was studied in primary cultures of human thymic epithelial cells by experimentally blocking the movement of secretory vesicles within these cells. These cultures were subjected to cytoskeleton inhibitors, such as colchicine and/or cytochalasin B, that block the directed movement of secretory vesicles, or to monensin, an ionophore that specifically perturbs the traffic of Golgi-derived vesicles. Both cytoskeleton inhibitors partially prevented thymulin secretion into the culture supernatants, and their effects were dose-dependent. Moreover, the percentage of thymulin-containing cells (evaluated by immunofluorescence with a zinc-specific anti-thymulin monoclonal antibody), as well as the fluorescence intensity within these cells, was significantly higher than observed in control cultures, suggesting that the hormone was accumulated in the cytoplasm, thus facilitating its detection. Similar results were obtained with monensin. These results, together with the recent identification of high molecular weight proteins reacting with anti-thymulin antibodies, suggest that thymulin is secreted via the following intracellular pathway: a precursor is synthesized at the level of the granular endoplasmic reticulum; it migrates to the Golgi complex, from which it is released within hormone-containing vesicles; the vesicles incorporate zinc, move towards the cell membrane, and eventually fuse with it. This sequence of events characterizes the classical phenomenon of exocytosis.