Glyoxysomes, a group of specialized peroxisomes, are organelles that degrade fatty acids by the combination of fatty acid beta-oxidation and glyoxylate cycle. However, the mechanism underlying the transport of the fatty acids across the peroxisomal membrane is still obscure in higher plant cells. We identified and analyzed the PED3 gene and its gene product, Ped3p. The phenotype of the Arabidopsis ped3 mutant indicated that the mutation in the PED3 gene inhibits the activity of fatty acid beta-oxidation. Ped3p is a 149-kDa protein that exists in peroxisomal membranes. The amino acid sequence of Ped3p had a typical characteristic for "full-size" ATP-binding cassette (ABC) transporter consisting of two transmembrane regions and two ATP-binding regions. This protein was divided into two parts, that had 32% identical amino acid sequences. Each part showed a significant sequence similarity with peroxisomal "half" ABC transporters so far identified in mammals and yeast. Ped3p may contribute to the transport of fatty acids and their derivatives across the peroxisomal membrane.