Despite the importance of peroxisomal oxidation in branched chain lipid (phytol, cholesterol) detoxification, little is known regarding factors regulating the peroxisomal uptake, targeting, and metabolism of these lipids. Although in vitro data suggest that sterol carrier protein-x (SCP-x) plays an important role in branched-chain lipid oxidation, the full physiological significance of this peroxisomal enzyme is not completely clear. To begin to resolve this issue, SCP-x null mice were generated by gene ablation of SCP-x from the SCP-x/SCP-2 gene and fed a phytol-enriched diet to characterize the effects of lipid overload in a system with minimal 2/3-oxoacyl-CoA thiolytic activity. It was shown that SCP-x gene ablation: (i) did not result in reduced expression of SCP-2 (previously thought to be derived in considerable part by post-translational cleavage of SCP-x); (ii) increased expression levels of key enzymes involved in - and -oxidation; and (iii) altered lipid distributions leading to decreased hepatic fatty acid and triglyceride levels. In response to dietary phytol, lack of SCP-x resulted in (i) accumulation of phytol metabolites despite substantial up-regulation of hepatic peroxisomal and mitochondrial enzymes; (ii) reduced body weight gain and fat tissue mass; and (iii) hepatic enlargement, increased mottling, and necrosis. In summary, the present work with SCP-x gene ablated mice demonstrates for the first time a direct physiologic relationship between lack of SCP-x and decreased ability to metabolize branched-chain lipids.