There is increasing evidence that the association of prostate carcinoma cells with the surrounding extracellular matrix is important for their growth. Proteoglycans (PGs) are components of matrix that form important associations with other molecules. Heparan sulfate proteoglycan (HSPG), for example, associates with several matrix components such as fibronectin, laminin, and basic fibroblast growth factor. The purpose of this study was to determine if human prostate carcinoma cells (PC-3) synthesized HSPG in tissue culture. Three areas of sulfate-labeled matrix were collected from PC-3 cells and analyzed: attachment sites, nonattachment cell surfaces, and media. Charged groups were separated by ion-exchange columns, and PGs identified by gel filtration chromatography after chemical and enzymatic treatment. The media fraction contained the greatest proportion of sulfated PGs (79.3%), of which 45.5% were HSPG. The greatest concentration of HSPG was in the attachment site fraction where 88.6% of PGs were HSPG, although only 11.7% of sulfated molecules were PGs. In nonattachment surfaces, only 19.6% were PGs, of which 19.4% were HSPG. When PGs were assessed for hydrophobic binding to octyl-Sepharose beads, only a proportion of HSPG in the media fraction contained hydrophobic domains (18.8%). In summary, PC-3 cells synthesize at least two types of HSPG, sorting them into different matrix compartments. The major PG in attachment sites is HSPG without a hydrophobic domain, while an HSPG in the media fraction has a hydrophobic domain. The localization of different types of HSPG may be functionally important and may be altered during the metastatic process for prostate carcinoma when in association with specific stroma.