Preincubation of cytolytic T lymphocytes (CTLs) generated in secondary C57BL/6 anti-DBA/2 mixed leukocyte cultures with an inhibitor of cellular cholesterol synthesis (25-OH-cholesterol) for 24 hr strongly depressed the cytolytic activity as determined in a 3-hr 51Cr assay. The effect of the inhibitor was reversed by the simultaneous addition of cholesterol or of mevalonic acid during the preincubation period (mevalonate is the product of the regulatory enzyme in the sterol synthesis pathway, 3-hydroxy-3-methylglutaryl-CoA reductase (NADP) [mevalonate:NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34]). Because, under the same culture conditions, inhibition of DNA synthesis had no effect on CTL activity, the experiments suggest that the effect of 25-OH-cholesterol is related to its inhibitory effect on sterol synthesis, resulting in decreased levels of membrane-bound cholesterol, rather than to inhibition of cellular proliferation.