In a study to employ progenitor-derived clonogenic assays of granulocyte-macrophage colony-forming units (CFU-GM) and erythroid colony-forming units (CFU-E) for the assessment of chemically induced hematotoxicity in rats, we sought the appropriate culture medium for each assay. Then, the effect of cyclophosphamide or phenylhydrazine on bone marrow cells was examined in vivo and in vitro. Oral treatment of rats with 25 mg/kg of cyclophosphamide significantly decreased the number of CFU-GM, which was an earlier and more sensitive index than were other hematological parameters tested. Direct exposure of the culture to cyclophosphamide had little effect on CFU-GM production, but addition of sera from rats pretreated with cyclophosphamide strongly depressed their formation, which suggested that the active metabolites of cyclophosphamide produced in the body may play an important role in toxicity. Subcutaneous injection of rats with 40 mg/kg of phenylhydrazine produced a marked increase in CFU-E 6-24 hr after injection. Direct exposure of the culture to phenylhydrazine had severe cytotoxic effects, but addition of sera from rats receiving phenylhydrazine resulted in increased numbers of CFU-E, probably indicative of increases in endogenous erythropoietin in donor rats. These results demonstrate that the proper use of progenitor assays in the in vivo and in vitro studies may be a valuable tool for approaching the mechanism underlying hematotoxicity.