The mechanisms of tumor cytotoxicity of rat polymorphonuclear leukocytes (PMN) activated with cytokine(s) were studied with the use of supernatants from a rat myelomonocytic leukemia cell line, WRT-7, incubated in the presence of bacterial lipopolysaccharide (LPS) (LPS WRT-7 sup) as a source of cytokine. Rat peritoneal PMN treated with LPS WRT-7 sup showed cytostasis from 3 hr after the start of incubation, while significant cytolysis was first observed after 24 hr. When target tumor cells were separated from PMN at 6 or 12 hr after the start of the assay, 3H-UdR release from the separated target cells comparable to that from the group incubated with PMN for the whole assay time of 40 hr was observed during the following incubation, which indicates that priming for subsequent lysis occurs at a relatively early stage of the assay. None of various scavengers of active oxygens, inhibitors of heme enzymes, and inhibitors of neutral proteinases inhibited cytolysis mediated by PMN stimulated with LPS WRT-7 sup. Heparin inhibited PMN cytolysis only when it was added within 1 hr after the start of the assay. Fractionation of heparin by ion exchange chromatography showed a parallelism between the negative charge and the inhibitory effect of heparin on PMN cytotoxicity.