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The mechanism of action of the cytokine, interleukin-l (IL-1), has been investigated. Mouse thymoma (EL4 6.1) cells were preincubated with [³H]-glycerol and then incubated with recombinant IL-1β for varying periods. Interleukin-1 caused a rapid increase in diacylglycerol production (approx. 2 fold at 30 sees). This reproducible enhancement of diacylglycerol accumulation was abolished by pretreatment of the cells with pertussis toxin. Interestingly, a similar IL-1 induced increase in diacylglycerol was observed when the cells were preincubated with [³H]-myristic acid. These results appear to suggest a novel mode of action of interleukin-1 which involves a G-protein mediated breakdown of a membrane lipid resulting in the production of diacylglycerol. It is suggested that one possible candidate for this parent lipid may be a phosphatidylinositol glycan.