Osteoclasts are derived from hemopoietic precursors in the marrow. Their differentiation pathway is still undefined, but an important role was observed for the marrow microenvironment in the regulation of osteoclasto-genesis. Various marrow stromal cell subtypes were used to study their possible role in the formation of osteoclasts from myeloblast (M1) cells. Interactions between M1 cells and the 14F1.1 endothelial-adipocyte stromal cell line were demonstrated in a coculture model. M1 cells attached to the adherent layer of 14F1.1 cells and formed distinct foci reminiscente of ''cobblestone areas.'' Following these interactions, M1 cells developed specific enzymatic activities and became multinucleated. Both mononuclear and multinuclear M1 cells became positive to tartrate-resistant acid phosphatase (TRaP) and ATPase, a feature characteristic of osteoclasts, and were also responsive to calcitonin. Furthermore, they attached to mineralized bone particles and their membrane changed into a ruffled border at the zone of interaction with the bone matrix. We thus demonstrated that marrow endothelial-adipocytes may play a role in regulating the differentiation of myeloblasts into osteoclasts. (C) 1994 Wiley-Liss, Inc.