Recent data suggest that impaired mitochondrial activities in Zellweger fibroblasts are related to defective peroxisome biogenesis and vice versa. To investigate the contribution of functional mitochondria to cholesterol biosynthesis, radioactive precursor molecules that form acetyl-CoA via beta-oxidation-independent (pyruvate) or -dependent (palmitate and octanoate) pathways were used. Production of both C-14-labeled cholesterol and C-14-labeled CO, from these radioactive tracers was significantly impaired in peroxisomal -defective ZR-82 Chinese hamster ovary cells in comparison to controls. In contrast, cholesterol synthesis from acetate-a tracer directly converted to acetyl-CoA without the involvement of mitochondrial activities-was threefold higher in ZR-82 cells than in controls. Pathways further contributing to cellular cholesterol homeostasis, i.e., receptor-mediated binding of exogenous lipoprotein-associated cholesterol as well as intracellular mobilization of cholesteryl ester deposits were similar in ZR-82 and controls. From these findings, we propose that peroxisomal dysfunction in ZR-82 cells is tightly coupled to impaired mitochondrial activities. e.a.. defective mitochondrial P-oxidation and formation of acetyl-CoA from short chain fatty acids resulting in a decreased rate of CO, production, and an apparent decrease in cholesterol biosynthesis. Actually, cholesterol biosynthesis from acetate is increased in the peroxisomal-defective cells. This explains previous conflicting conclusions. (C) 2003 Elsevier Science (USA). All rights reserved.