Cultured human lung fibroblasts produce a large, nonhydrophobic heparan sulfate proteoglycan that accumulates in the extracellular matrix of the monolayer (Heremans, A., J. J. Cassiman, H. Van den Berghe, and G. David. 1988. J. Biol. Chem. 263: 4731-4739). A panel of four monoclonal antibodies, specific for four distinct epitopes on the 400-kD core protein of this extracellular matrix heparan sulfate proteoglycan, detects similar proteoglycans in human epithelial cell cultures. Immunohistochemistry of human tissues with the monoclonal antibodies reveals that these proteoglycans are concentrated at cell-matrix interfaces. Immunogold labeling of ultracryosections of human skin indicates that the proteoglycan epitopes are nonhomogeneously distributed over the width of the basement membrane. Immunochemical investigations and amino acid sequence analysis indicate that the proteoglycan from the fibroblast matrix shares several structural features with the large, low density heparan sulfate proteoglycan isolated from the Engelbreth-Holm-Swarm sarcoma. Thus, both epithelial cell sheets and individual mesenchymal cells accumulate a large heparan sulfate proteoglycan(s) at the interface with the interstitial matrix, where the proteoglycan may adopt a specific topological orientation with respect to this matrix.