Normal adult C57BL, BALB/c, and C3H/HeJ mice were injected intraperitoneally three times daily for up to 6 days with 102,000 U (200 ng) per injection of purified, bacterially synthesized, Multipotential colony-stimulating factor (CSF) (Interleukin-3) (rMulti-CSF) and compared with control mice injected with serum/saline with or without added endotoxin (1 ng/mL). Mice injected with rMulti-CSF exhibited tenfold rises in blood eosinophil and twofold to threefold rises in neutrophil and monocyte levels. The spleens from mice injected with rMulti-CSF showed a 50% increase in weight, elevated levels of maturing granulocytes, eosinophils, nucleated erythroid cells and megakaryocytes, and up to 100-fold rises in mast cells. Progenitor cell frequencies in the spleen were elevated sixfold to 18-fold. No significant changes were observed in the marrow. Sixfold to 15-fold rises were observed in peritoneal cell populations of mice injected with rMulti-CSF with evidence of increased peritoneal macrophage phagocytic activity. Livers of C57BL mice, but not of the other strains, exhibited increased numbers of infiltrating hematopoietic cells whereas rises in mast cell numbers were observed in the mesenteric lymph node, skin, and gut in BALB/c and C3H/HeJ mice. Endotoxin was excluded as being responsible for the observed changes except possibly those involving peritoneal macrophage phagocytic activity. The results indicate that the injection of normal mice with rMulti-CSF significantly stimulates the same types of hematopoietic populations as are stimulated in vitro by Multi-CSF and indicate that this and other CSFs should be useful in stimulating hematopoietic repopulation and functional activity in vivo.