Peritoneal macrophages (M phi) from C3H/HeN mice became cytotoxic for 1023 tumor cells after incubation with lymphokine (LK) for 8-12 hr and lost tumoricidal activity by 22 hr in the continued presence of LK; bacterial endotoxin (LPS) was ineffective in inducing tumoricidal activity. M phi from C3H/HeJ mice were not activated for tumor cytotoxicity after treatment with LK or LPS. C3H/HeN M phi acquisition of tumoricidal activity was accompanied by unique changes in M phi lipid composition: cellular content of cholesterol (CHOL) and polyunsaturated fatty acids (UFA) increased two- to threefold when the cells showed maximal tumoricidal activity and returned to control levels when the M phi lost tumoricidal activity. LPS treatment of C3H/HeN M phi and LK or LPS treatment of C3H/HeJ M phi did not cause characteristic M phi lipid alterations. To determine at what stage during M phi activation the correlative CHOL and UFA compositional changes were occurring, C3H/HeN M phi were primed with LPS or low concentrations of LK and triggered with LPS or Lk; M phi lipid and fatty acid composition was monitored at each stage. LK was shown to be able to prime and trigger whereas LPS could only trigger LK-primed M phi for tumor cytotoxicity. In all cases, the increase in M phi CHOL and UFA content occurred at the triggering step for tumor cytotoxicity rather than at the priming step. These data suggest that there is a correlation between the effects of endogenous and exogenous factors that control expression of M phi tumoricidal activity and their effects on M phi CHOL and UFA content; the establishment of these changes in M phi lipid composition occurs at a time when the cells are triggered for tumor cytotoxicity.