In the mouse hematopoietic system, 5-fluorouracil (5-FU) reversibly inhibits the generation of multilineage colonies containing granulocyte, erythroid, megakaryocyte, and macrophage lineage. To determine the effect of 5-FU on mastopoiesis in vitro, bone marrow cells were obtained from mice, cultured, and treated with 5-FU for 14 days in an interleukin-3 (IL-3)-enriched medium. A dose-related inhibitory effect of 5-FU on mastopoiesis was found. When an inhibitory dose (1 microgram/mL) of 5-FU was supplemented to the cultures for only 2, 4, or 8 days and the cells were then recultured without the drug, we observed inhibition of mastopoiesis directly related to the time of exposure of the cells to 5-FU. To determine the effect of 5-FU on mastopoiesis in vivo, bone marrow cells from mice that had received a single intravenous (i.v.) 5-FU injection (150 mg/kg) were cultured. A virtually total absence of mast cells was noted at days 1 and 2 following 5-FU administration. A gradual reappearance of mast cells was later observed. Whether mice were injected with the drug once or with four once-daily (100 mg/kg 5-FU) injections, a similar pattern of delay of mast cell appearance was observed. The findings suggest (1) an irreversible, nonadditive, toxic effect of 5-FU on mast cell precursors and (2) that most or all of the mast cell precursors are nonquiescent cells, continuously activated or cycling. In addition, the use of 5-FU may serve as a unique model system for controlling and studying mastopoiesis in normal mice, rather than the mutated mice currently studied.