We used an established Drosophila cell line (Kc cells), which neither synthesized nor required cholesterol for growth, to determine if sterol and nonsterol modulators of vertebrate 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity were also active in this biological system. Drosophila HMG-CoA reductase was membrane-bound and required NADPH for catalysis. In contrast to the vertebrate enzyme, Kc cell HMG-CoA reductase activity was not modulated by cholesterol (10 micrograms/ml), human low density lipoprotein (83 micrograms of cholesterol/ml), or oxygenated sterols (5-10 micrograms/ml). However, mevalonate caused a rapid strong suppression of Kc HMG-CoA reductase activity; 18 microM R-mevalonate produced 50% suppression of the enzyme within 24 h. Compactin, a competitive inhibitor, decreased HMG-CoA reductase activity in Drosophila embryo cell-free extracts with an apparent Ki of 1.0 nM. Kc cells, grown in the presence of compactin, had a HMG-CoA reductase specific activity 5- to 10-fold higher than untreated cells. Mevalonate blocked this increase. We have concluded that HMG-CoA reductase activity in Kc cells is (a) not responsive to feedback inhibition by sterols, and (b) is controlled by a fundamental sterol-independent regulatory process. The signal for modulation of HMG-CoA reductase activity may be mevalonate and/or its magnitude conversion to a nonsterol isopentenoid precursor and/or end product. These observations may have broader validity, not only for other insect cells, but for eukaryotic cells in general.