Peritoneal macrophages (M phi) from mice became cytotoxic after incubation with lymphokine (LK); tumoricidal activity was evident with M phi treated with LK for 4 hr, became maximal after 8-12 hr of incubation, and decreased to control levels by 24-36 hr. LK induced marked changes in M phi lipid composition: cellular content of cholesterol (CHOL) and polyunsaturated fatty acid (UFA) content of cellular lipids (especially 18:3) increased two- to threefold after 8 hr when the cells showed maximal tumoricidal activity. Cellular lipid and fatty-acid content returned to control levels by 24 hr when the M phi had lost tumoricidal activity. These changes were not observed with equal numbers of M phi cultured in control supernatants. To analyze the role of CHOL and UFA in M phi tumor cytotoxicity, casein-induced peritoneal M phi were enriched in CHOL or linolenic acid (18:3) and then tested for their ability to kill 1023 tumor cells. The 18:3-enriched cells were markedly tumoricidal, whereas controls cultured in delipidized medium alone or enriched with saturated fatty acid (18:0) were not cytotoxic. CHOL-enriched M phi were not tumoricidal; indeed, these cells were inhibited in their killing after treatment with LK compared to M phi cultured in delipidized medium with LK alone. The effects of 18:3 and CHOL enrichment of the M phi on their metabolic status, inflammatory function, and tumor cell-binding capacity were tested. The 18:3-enriched M phi were depressed in their ability to synthesize protein and in phagocytic activity compared to controls; these cells showed a transient increase in superoxide release. M phi cultured with 18:3 for 48 hr were also cytotoxic for P815 tumor cells, but did not show an enhanced capacity for P815 binding compared to controls. CHOL-enriched M phi were similar to control cells in their protein synthesizing and phagocytic activities; these cells also showed an early transient increase in superoxide release. CHOL-enriched M phi were not cytotoxic for P815 cells, but bound the tumor cells more readily than did the 18:3-enriched M phi. The data suggest that endogenous levels of 18:3 and CHOL can regulate M phi tumor cytotoxicity, but not through regulation of M phi protein synthesis, oxidative metabolism, or augmented capacity for tumor target binding.