We have generated a line of mice deficient in adrenoleukodystrophy protein (ALDP) by gene targeting in order to clarify the pathophysiology of adrenoleukodystrophy (ALD). ALDP-deficient male and female mice appeared normal clinically at least up to 12 months. Western blot analysis showed the absence of ALDP in the brain, spinal cord, lung, and kidney and normal expression of PMP70 in the liver, lung, and kidney. The amounts of C26:0 increased by 73-240% in the brain, spinal cord, lung, and kidney. beta-Oxidation of very long chain fatty acids (VLCFA) in cultured hepatocytes and fibroblasts was reduced to 35-50% of normal. Light and electron microscopy did not show demyelination in the brain, spinal cord, and peripheral nerve. Thus, the deficiency of ALDP in mice impairs the peroxisomal fatty acid beta-oxidation but does not duplicate the clinical and pathological abnormalities of the human ALD. These observations suggest that the accumulation of VLCFA alone is not sufficient to cause demyelination in the nervous system.