Very long chain fatty acid (VLCFA) metabolism in astrocytes is important for the maintenance of myelin structure in central nervous system. To analyze the contribution of the ABCD1-dependent and -independent pathways to VLCFA metabolism in astrocytes, we prepared human glioblastoma U87 cells with a silencing of ABCD1 and primary astrocytes from abcd1-deficient mice, and measured fatty acid β-oxidation in the presence or absence of a potent inhibitor of carnitine palmitoyltransferase I, 2-[5-(4-chlorophenyl)pentyl]oxirane-2-carboxylate (POCA). In U87 cells, C24:0 β-oxidation was decreased to ca. 70% of the control in the presence of POCA, and the activity was further decreased to ca. 20% by the silencing of ABCD1. In mouse primary astrocytes, C24:0 β-oxidation was also decreased to ca. 70% of the control in the presence of POCA. The C24:0 β-oxidation in Abcd1-deficient primary astrocytes was ca. 60% of the wild-type cells and the activity was further decreased to ca. 25% in the presence of POCA. Compared to human skin fibroblasts, in which VLCFA β-oxidation is not significantly inhibited by POCA, approximately one-third of the overall VLCFA β-oxidation was inhibited in both types of astrocytic cells. These results suggest that VLCFA is indeed β-oxidized in ABCD1-dependent pathway, but the ABCD1-independent peroxisomal and mitochondrial β-oxidation pathways significantly contribute to VLCFA β-oxidation in astrocytic cells.Key words very long chain fatty acid β-oxidation; astrocyte; ABCD1; peroxisome; mitochondria Peroxisomes are single membrane organelles that are present in almost all eukaryotic cells. The peroxisomes are involved in a variety of metabolic processes, including the β-oxidation of fatty acids, especially very long chain fatty acids (VLCFA, >C22), and the synthesis of ether phospholipids and bile acids in mammals.1) Transport of substrates for fatty acid β-oxidation across the peroxisomal membrane is an essential step in this metabolism. Peroxisomal ATP-binding cassette (ABC) proteins have been implicated in this transport.To date, three ABC proteins, classified into "subfamily D," have been identified in mammalian peroxisomes. These are adrenoleukodystrophy protein (ALDP/ABCD1),2) ALDP-related protein (ALDRP/ABCD2) 3) and a 70-kDa peroxisomal membrane protein (PMP70/ABCD3). 4) Dysfunction of ABCD1 causes the human genetic disorder X-linked adrenoleukodystrophy (X-ALD) characterized by an accumulation of VLCFA because of the impaired peroxisomal β-oxidation of VLCFA.5) VLCFA β-oxidation in X-ALD patient fibroblasts was restored by the expression of ABCD1. Likewise, the expression of ABCD2, which has a high sequence similarity to ABCD1, also restored VLCFA β-oxidation in X-ALD fibro blasts.6) In analogy to the role of Pxa1p/Pxa2p, 7) yeast peroxisomal half-ABC transporters, which is involved in the transport of acyl-CoA, ABCD1 and ABCD2 are thought to be involved in the metabolic transport of VLCFA-CoA. 8,9) ABCD3 is suggested to be involved in the metabolic transport of long chain fatty acids (LCFA)...