Heparan sulfate (HS) present on the surface of hemopoietic stromal cells has important roles in the control of adhesion and growth of hemopoietic stem and progenitor cells. Recent studies have characterized several different heparan sulfate proteoglycans (HSPGs) from both human and murine bone marrow stromal cells. In the present study, we have compared the molecular structure of HS, metabolically labeled with [(35)S]-sulfate produced by two distinct preparations of murine hemopoietic stromal cell lines. These comprised a bone marrow-derived cell line S17 and a fetal liver-derived cell line AFT024. [(35)S]-HS was examined in the cell layers and in the culture medium. We identified and measured the relative proportions of the various glycosaminoglycans (GAGs) in the two stromal cell lines. Chondroitin sulfate (CS) was preponderantly secreted by the stromal cell lines, while HS was relatively more abundant in the cell-associated fractions. The two types of stromal cells differ in their HS composition, mainly due to different patterns of N- and O-sulfation. The two stromal cell lines expressed mRNA for different HSPGs. Data from reverse transcription PCR revealed that the two stromal cell lines expressed mRNA for glypican and syndecan4. Only AFT024 cell line expressed mRNA for betaglycan. There was no evidence for expression of mRNA for both syndecan1 and syndecan2. [(35)S]-sulfated macromolecules could be released from the cell surface of both stromal cell lines by phosphatidylinositol phospholipase C (PI-PLC), which is consistent with the expression of glypican detected by PCR experiments.