Staphylococcus aureus is an invasive pathogen capable of causing life-threatening disease. A major component of this pathogen's virulence is its ability to invade normal endovascular tissue. We examined the interaction of S. aureus with cultured human endothelial cells and with human and rabbit endovascular tissue. Our ultrastructural study demonstrated a sequence of steps which occurred with staphylococcal invasion of human endothelial cells; adhesion, endocytosis, and intracellular replication. Ultimately, this resulted in cell disruption and death. Cytochemical staining of lysosomes demonstrated lysosomal fusion with both viable and killed intracellular bacteria without evidence of staphylococcal degradation. Quantitative studies using an in vitro infection assay demonstrated comparable rates of adhesion by viable and ultraviolet-killed bacteria, phagocytosis at a slower rate, and intracellular replication. The present study demonstrates an active role for the endothelial cell in the development and spread of endovascular staphylococcal infections. It also supports the use of this in vitro tissue culture system as a model for the study of bacterial invasion of the endothelium.