The bone marrow microenvironment, which is composed of fibroblasts, endothelial cells, adipocytes and macrophages, plays an important role in the haematopoiesis and lymphopoiesis by producing various cytokines. Therefore, investigation of the origin of these cells following allogeneic bone marrow transplantation (BMT) is very significant, in terms of the haematological and immunological reconstitution after BMT. We have investigated the origin of fibroblastic stromal cells in long-term cultures in seven of the sex-mismatched cases. This was carried out by in situ hybridization using a Y-chromosome specific cDNA probe (PHY10), conserving the morphology of the cells. In situ hybridization analysis showed that bone marrow fibroblasts (BMF) in long-term cultures in all the sex-mismatched cases originated from the recipients. We have also performed Southern blot analysis using a PHY10 probe in the sex-mismatched cases and using a variable number of tandem repeats (VNTR) probe, which can detect DNA polymorphisms, in two of the sex-matched cases. In addition, we have employed polymerase chain reaction (PCR) using the VNTR marker (MCT118). Although all the patients showed haemopoietic engraftment with donor cells, their BMF were found, by Southern blot analysis and PCR method, to be of the recipient origin. These data indicate that bone marrow-derived fibroblastic stromal cells which proliferate in long-term cultures are not transplantable in the conditioning regimens used for allogeneic BMT in humans.