The effect of different fatty acids on tumour necrosis factor (TNF)-induced cytolysis of TNF-sensitive WEHI 164 murine fibrosarcoma cells has been determined. The saturated fatty acids palmitic (16:0) and stearic (18:0) acid strongly potentiated the cytolytic activity of TNF. They also had a slight cytotoxic effect in the absence of TNF. Contrary to this, the unsaturated fatty acids oleic (18:1), linoleic (18:2), alpha-linolenic (18:3), arachidonic (20:4), and eicosapentaenoic (20:5) acid were neither cytotoxic by themselves, nor did they have any significant effect on the cytolytic activity of TNF. Addition of either of the unsaturated fatty acids 18:2 or 20:5 together with 18:0 eliminated the potentiating effect 18:0 by itself had on TNF-induced cytolysis. A mixture of fatty acids resembling that found in cell phospholipids had no cytotoxic effect by itself, nor any effect on the cytolytic activity of TNF. The potentiating effect of saturated fatty acids on TNF thus seems to depend on their relative amount compared to the amount of unsaturated acids. The results indicate that TNF does not exert its cytotoxic effect simply by suppressing hydrolysis of triglyceride, thereby inducing an insufficient cellular supply of fatty acids.