Heparin is a natural anticoagulant molecule that can alter the activity and/or levels of other molecules involved in blood coagulation. Protein S, an anticoagulant protein, is synthesized and released into the plasma by endothelial cells. Immunological assays revealed a significant increase in protein S found in the media and in the endothelial cells after heparin treatment. Time assays revealed a rapid heparin effect on protein S levels in the media. Upon treatment with chondroitin sulfate, a small increase in the amount of protein S in the conditioned medium was also detected but the change in the cell-associated protein S levels after chondroitin sulfate treatment was a decrease rather than the increase implying that heparin and chondroitin sulfate are operating through different mechanisms. Radioimmunoprecipitations and cycloheximide treatments indicated no significant difference in protein S synthesis in heparin treated cells. In experiments comparing heparin and ammonium chloride effects, heparin seems to mimic the ammonium chloride effect on the levels of protein S in the media. Together, the data indicate that heparin increases the levels of protein S found in the media of cultured endothelial cells by producing a specific block in protein S degradation.