We investigated the influence of heparin on the composition of the subendothelial matrix and on endothelial permeability to elucidate the structure-function relationship of matrix composition and permeability. Albumin flux across the confluent endothelial monolayers was used to assess the macromolecular permeability. Lowest values were obtained when 100% serum was used as medium for permeability studies. The endothelial matrix components, fibronectin and basement membrane-associated heparan sulfate proteoglycan (HSPG), were measured by enzyme immunoassay. Treatment of proliferating endothelial cells with heparin (0-900 micrograms/ml) induced a dose-dependent decrease in endothelial HSPG content, whereas the fibronectin content was unaltered. This structural change was accompanied by an increase in albumin permeability. Both heparin effects exhibited similar dose-response curves with half-maximal effects at approximately 5 micrograms/ml heparin. Acute addition of 300 micrograms/ml heparin had no effect on permeability or HSPG content. When endothelial cells were preincubated with an HSPG antiserum, the endothelial permeability increased nearly threefold. Our results indicate that heparin-induced loss of HSPG may cause the increase in endothelial permeability. The data underline the importance of HSPG for the integrity of the endothelial barrier.