Hairy-cell leukemia (HCL) is a proliferation of clonal B-lymphocytes with features of activation. The disease has a number of distinctive characteristics, prominent among which is the fine reticulin fibrosis invariably present in the bone marrow. However, fibroblast infiltration has never been noted in the marrow and the origin of the fibrosis has not been established. The present studies show that the hairy cells (HCs) of HCL produce an insoluble matrix of fibronectin (FN) in vitro. FN synthesis was shown by the appearance of cellular FN on the surface of cells cultured in serum-free medium and by immunoprecipitation of the metabolically labeled protein from HC aggregates. Moreover, the HCs were shown to assemble FN into disulphide-bonded multimers. This assembly was blocked by a 70-kD amino-terminal fragment of the molecule that blocks FN multimer formation by fibroblasts. HCs expressed abundant VLA-5, an FN receptor not present on normal circulating B lymphocytes, but important in matrix formation. Furthermore, HCs were shown to adhere to an FN fragment containing the VLA-5 binding site. It is therefore suggested that the VLA-5 of HCs is implicated in their assembly of FN matrix. The in vivo relevance of the findings was established by the demonstration of FN in association with infiltrating HCs in bone marrow sections from patients with HCL. It is concluded that the HCs synthesise and assemble an FN matrix and that this is at least partly responsible for the bone marrow fibrosis so characteristic of the disease.