Endocytosis of horseradish peroxidase (HRP) was studied in primary cultures of cerebral endothelial cells prepared from 2-week-old rats. These cultures were considered "endothelial-like" on the basis of their ability to internalize acetylated low density lipoprotein. Cellular localization of HRP protein was examined at the light and ultrastructural levels and endocytosis of the protein was evaluated by a colorimetric assay. HRP was localized in discrete cytoplasmic granules by light microscopy. At the ultrastructural level these granules corresponded to pleomorphic membrane-bound structures that were present throughout the cytoplasm. The amount of internalized HRP was directly related to the concentration of the protein in the medium, was not saturable at high concentrations of HRP, and increased with time. Endocytosis proceeded at 37 degrees C, but was abolished at 4 degrees C. In pulse-chase experiments, the quantity of internalized protein in the cells did not significantly change during the 2 hr chase period. Taken together, these findings suggest that internalization of HRP occurs by fluid-phase endocytosis, a non-receptor-mediated process, and that the protein is stable within an intracellular compartment for at least several hours.