Polymorphonuclear leukocytes (PMNs) recovered from the peritoneal cavity of mice treated with the streptococcal preparation OK-432, exhibited strong cytotoxicity after the in vitro addition of Nocardia rubra cell wall skeleton (N-CWS). In this study, we investigated whether recombinant human granulocyte colony-stimulating factor (rhG-CSF) could augment the cytotoxicity of OK-432-induced PMNs after the addition of N-CWS in vitro. PMNs recovered from the peritoneal cavity of 8- to 10-week-old, male C3H/He mice induced by intraperitoneal (i.p.) injection of 50 KE/kg (1 KE = 0.1 mg) of OK-432 were used in a 51Cr release assay against MM46 mammary carcinoma cells. While addition of rhG-CSF in vitro did not augment the cytotoxicity of OK-432-induced PMNs, marked augmentation of the cytotoxicity of OK-432-induced PMNs was observed following a single subcutaneous (s.c.) or i.p. injection of 125 micrograms/kg of rhG-CSF. The effect of in vivo administered rhG-CSF was dependent on the timing of the injection with respect to OK-432 administration and differed from s.c. or i.p. injections. Interestingly, the cytotoxicity of OK-432-induced PMNs was rather weak following consecutive s.c. or i.p. administration of rhG-CSF for 7-14 days. H2O2 is likely involved in mediating the cytotoxicity of OK-432-induced PMNs since activity was significantly reduced by the in vitro addition of low concentration of catalase. Generation of H2O2 by the PMNs correlated with cytotoxicity. These results suggest that in vivo administration of rhG-CSF augments the cytotoxicity of OK-432-induced PMNs in a time dependent fashion and that H2O2 plays an important role in mediating their cytotoxicity.