Syndecan-1 (CD138) mediates myeloma cell adhesion, and loss of syndecan-1 from the cell surface may contribute to myeloma proliferation and dissemination. Flow cytometry analysis of myeloma cells in bone marrow specimens shows heterogeneity in cell surface syndecan-1 expression. It is not known whether weaker expression correlates with more aggressive disease. However, recent reports suggest that variations in syndecan-1 staining intensity on myeloma cells may be an artifact of specimen handling. In this study, we evaluate syndecan-1 expression in bone marrow biopsy sections from 28 multiple myeloma patients, to elucidate the heterogeneity of syndecan-1 expression in situ. Immunoreactivity for syndecan-1, using the antibody B-B4 (CD138), was found in more than 95% of multiple myeloma cells in 27 of 28 biopsies. However, one biopsy had more than 50% CD138-negative cells and cells with weak CD138 expression were identified in the majority of cases. Loss of syndecan-1 did not appear to relate to myeloma cell differentiation. In addition, syndecan-1 was detected on intravascular and intrasinusoidal myeloma cells suggesting that loss of syndecan-1 may not be required for extramedullary dissemination. Bone marrow biopsies from nine additional patients, with variable CD138 staining intensity on myeloma cells as determined by flow cytometry, were studied by immunohistochemistry. The heterogeneous CD138 expression was confirmed in situ, with weakly positive cells concentrated in areas of reticulin fibrosis. These cells had a disrupted pattern of membrane staining in contrast to the strong linear membrane staining seen in the other multiple myeloma cells. In addition, the fibrotic stroma stained intensely for syndecan-1. Accumulation of syndecan-1 within the extracellular matrix of the marrow likely is derived by shedding of the molecule from the surface of myeloma cells. Because syndecan-1 can act to regulate the activity of heparan-binding growth factors, these reservoirs of syndecan-1 may play a critical role in promoting myeloma pathogenesis, or in regeneration of the tumor after chemotherapy.