Cytochromes P450 are a superfamily of proteins [1] which are involved in the oxidative metabolism of both foreign and endogenous compounds [2]. The cytochrome P450 4A family is known to be highly induced by peroxisome proliferators in mouse liver [3,4], although there is constitutive expression of one gene [5]. The CYP4A [6], CYP4B [7,8] and CYP4F [9,10] proteins are known to have fatty acid hydroxylase activity, and there is extensive speculation that the formation of hydroxylated fatty acids by cytochrome P450 leads to the production of physiologically active metabolites that regulate physiological function [11][12][13][14][15][16].Cytochrome P450 metabolism of fatty acids may also be of fundamental importance in brain [17][18][19], and it is known that neurotransmitters and fatty acids can be actively metabolized by cytochrome P450 in brain [18,20,21]. Although the specific content of cytochrome P450 in brain is relatively low compared with liver [17,22,23], this level of cytochrome P450 can be induced by various agents [24]. A peculiar feature of brain P450 is that it is difficult to account for the total P450 content with previously characterized P450 proteins [17].We describe the cloning of human and mouse cDNAs for the CYP4x1 P450, a molecular model of the protein, and tissue-specific localization of the RNA in mouse and human. The Cyp4x1 protein was localized by immunohistochemistry and shown to be a major P450 protein in mouse brain. A novel cytochrome P450, CYP4x1, was identified in EST databases on the basis of similarity to a conserved region in the C-helix of the CYP4A family. The human and mouse CYP4x1 cDNAs were cloned and found to encode putative cytochrome P450 proteins. Molecular modelling of CYP4x1 predicted an unusual substrate binding channel for the CYP4 family. Expression of human CYP4x1 was detected in brain by EST analysis, and in aorta by northern blotting. The mouse cDNA was used to demonstrate that the Cyp4x RNA was expressed principally in brain, and at much lower levels in liver; hepatic levels of the Cyp4x1 RNA were not affected by treatment with the inducing agents phenobarbital, dioxin, dexamethasone or ciprofibrate, nor were the levels affected in PPARa-⁄ -mice. A specific antibody for Cyp4x1 was developed, and shown to detect Cyp4x1 in brain; quantitation of the Cyp4x1 protein in brain demonstrated 10 ng of Cyp4x1 proteinAEmg )1 microsomal protein, showing that Cyp4x1 is a major brain P450. Immunohistochemical localization of the Cyp4x1 protein in brain showed specific staining of neurons, choroids epithelial cells and vascular endothelial cells. These data suggest an important role for Cyp4x1 in the brain.Abbreviations DAB, 3,3¢-diaminobenzidine; TCDD, 2,3,7,8 tetrachlorodibenzo-p-dioxin.