We investigated whether recombinant human granulocyte colony-stimulating factor (rhG-CSF) enhanced the cytotoxicity of PSK-induced polymorphonuclear leukocytes (PMNs) in the peritoneal cavity. Male C3H/He mice, 8- to 10-week-old, received single subcutaneous (s.c.) or intraperitoneal (i.p.) injection of 2.5 micrograms/animal of rhG-CSF at different time points before or after an i.p. administration of PSK. In other experiments, mice were s.c. or i.p. treated with the same dosage of rhG-CSF every day for 7 or 14 consecutive days and i.p. injected with 2.5 mg/animal of PSK on the last day. Peritoneal PMNs were harvested 6 hrs after the administration of PSK and purified to more than 95% by Ficoll-Paque for in vitro cytotoxic assay. In vitro cytotoxic assays with 51Cr labeled MM46 mammary carcinoma cells were added with 5-20 micrograms/ml of Nocardia rubra cell wall skeleton (N-CWS) at the beginning of the assay to augment the cytotoxic activity of PMNs. In vitro addition of rhG-CSF to the assay did not enhance the cytotoxicity of PSK-induced PMNs. However, the cytotoxicity was significantly increased when rhG-CSF was s.c. administered 12 hrs before a PSK injection or 2 or 5 hrs after that. On the other hand, the cytotoxicity was rather weak when mice s.c. or i.p. received consecutive injections of rhG-CSF. This cytotoxicity may be mediated by H2O2, since H2O2 production of PMNs during the cytotoxic assay appears to correlate with the levels of cytotoxicity under suppressed H2O2 generation by catalase or enhanced generation by rhG-CSF. These results suggest that rhG-CSF augments the cytotoxicity of PSK-induced PMNs when administered in vivo timely.