We have compared four assays to detect hemopoietic stromal damage induced by various cytostatic agents in young (4-week old) and adult (12-week old) mice. These assays included: (a) quantitation of the hemopoietic stem cell content of subcutaneously implanted spleens and femurs, (b) quantitation of fibroblastic colony-forming units per femur and spleen, (c) quantitation of the growth of normal hemopoietic progenitor cells in irradiated cytostatic drug-treated mice, and (d) measurement of splenic hemopoietic stem cell accumulation in response to bacterial lipopolysaccharide-induced hemopoietic stress. Busulfan caused a short- and long-term hemopoietic stromal defect. However, the four assays used showed different kinetics and severity of the stromal damage. Cyclophosphamide treatment resulted in a short-term stromal damage which was repaired within one week to three months, depending on the assay used. Methotrexate and vincristine did not cause long-term stromal damage as measured by the four assays used, whereas a short-term splenic stromal damage was detected using the subcutaneous implantation technique. No significant differences in stromal sensitivity to drug treatment were observed between young and adult mice. The presented data suggest that the four assays used to study stromal integrity measure different components of the hemopoietic microenvironment, and indicate that the use of a single assay may well lead to erroneous interpretations.