ABC transporters are members of a superfamily of membrane proteins involved in the transport of a variety of molecules across biological membranes. PMP70 is one of four ABC transporters in the mammalian peroxisome membrane; others are ALDP, ALDP-related (ALDR) and PMP70-related (PMP70R). Mutations in ALD result in accumulation of VLCFA due to impaired β-oxidation in peroxisomes and cause X-linked adrenoleukodystrophy. The function of PMP70 is unknown although PMP70 mutations have been observed in 2 patients with Zellweger syndrome. To understand its function and relationship to human disease, we targeted the murine PMP70 gene and produced PMP70 knockout mice. Products of heterozygous matings were born in expected Mendelian ratios. The knockout pups show a slower growth rate (70%) than that of wild type littermates for the first month of life but eventually catch up. PMP70 is absent by Northern and immunoblot analysis. Immunohistochemistry and electron microscopy show that peroxisomes are present and contain normal amounts of PTS1 and PTS2 targeted matrix proteins. β-oxidation of monocarboxylic fatty acid in PMP70-/- fibroblasts is normal. In liver, the peroxisome number appears to be increased approximately 2-fold. Metabolic studies in the PMP70-/- mice show a striking dicarboxylic aciduria. This result is particularly interesting because oxidation of dicarboxylic acids occurs mainly in peroxisomes. To explain our results, we suggest that PMP70 function involves transport of dicarboxylic acids into peroxisomes. In the absence of this transport, peroxisome β-oxidation of dicarboxylic acids is reduced; these compounds accumulate and cause peroxisome proliferation.