Pentosan polysulfate (PPS) has been shown to improve symptoms of patients with osteoarthritis (OA) when studied under double-blinded conditions. Laboratory studies indicated that this drug exhibits multiple actions, including the preservation of articular cartilage (AC) proteoglycans in animal models of OA and the stimulation of hyaluronan synthesis by synovial fibroblasts in vitro and in vivo. As PPS is strongly anionic and has a molecular weight of ∼5700 Da its ability to enter connective tissues rich in proteoglycans and interact with the resident cells has been questioned. In the present studies, experiments were undertaken to isolate and characterize proteins in human AC which have the potential to bind PPS. Thrombospondin was identified in 4.0 m GuHCl extracts of human AC as a PPS-binding protein. Furthermore, synovial fibroblasts derived from OA joints were shown to secrete thrombospondin and also bind PPS. Using bovine erythrocytes conjugated with PPS a rosetting of the synovial fibroblast could be demonstrated. The level of rosetting was not affected by pre-incubating cultures with thrombospondin antibody suggesting that PPS was interacting directly with the cells. Kinetic studies of 3 H-PPS uptake by synovial fibroblasts showed saturation of binding sites within 30 min when cells were maintained at 4°C but preservation of drug uptake for up to 120 min when cells were cultured at 37°C. These data, together with the finding that cells labeled with drug at 37°C showed higher incorporation, than at 4°C after trypsin digestion suggests that PPS first binds to the cell membrane then at 37°C is internalized, possibly by pinocytosis.