A mutant requiring both cholesterol and oleate for growth has been isolated from mutagenized Chinese hamster ovary cells. By comparison with wild-type cells, sterol and unsaturated fatty acid biosynthetic activities in the mutant cells grown in fetal calf serum medium appear to be nearly intact. However, whole-cell radioactive acetate, mevalonate, dihydrolanosterol, and stearate incorporation studies show that sterol synthesis from acetate, lanosterol demethylation, and fatty acid desaturation are defective in the mutant cells grown in delipidated serum medium. In vitro enzyme assays with crude cell extracts demonstrated that beta-hydroxy-beta-methylglutaryl-coenzyme A reductase is not induced in the mutant. These experiments were substantiated by gas/liquid chromatographic analyses which showed the sterol content and the percentage unsaturated fatty acids in mutant cells to be drastically reduced when the cells are grown in delipidated serum medium. A spontaneous revertant exhibiting prototrophic growth in lipid-free medium has been isolated from 50 X 10(6) mutant cells. All three defects in this revertant reverted back in parallel, which suggests that these three biosynthetic activities may be controlled by a common regulatory mechanism.