The toxicity of antifolate drug therapy on microenvironmental function of hemopoietic marrow stromal cells may be critical enough to justify the development of a simple cell culture model to appraise the dihydrofolate reductase (DHFR)/tetrahydrofolate dehydrogenase (EC 1.5.1.3) (4HFDH) enzymic system in individual stromal cells. The localization and pattern of expression of 4HFDH/DHFR in cultured bone marrow stromal cells were studied in situ in morphologically identifiable cells. The 4HFDH/DHFR expression varies quantitatively and qualitatively according to cell types and thus constitutes specific phenotypic patterns for fibroblastic stromal cells, endothelial cells, monocyte-macrophages, and osteoclast-like cells. Data indicate a good correlation between the labeling of the enzymic expression and the resistance of cells to methotrexate treatment. This conclusion is also supported by similar features shown by cell sublines bearing multiple DHFR gene copies. Levels of 4HFDH/DHFR expression in marrow stromal cells should provide useful markers underlining the effects of cytostatic drugs used in chemotherapy (for example, methotrexate injuries or resistance). The method can be applied to isolated cells, stromal colonies (fibroblast CFU), or whole adherent layers from marrow cultures. Moreover, with this method, enzymic reactions can be carried out in situ and visual correlations between a supportive microenvironment (hemopoietic foci) and the 4HFDH/DHFR levels on the stromal adherent layer are possible.