Autoantibodies (aAbs) are generated by the immune system in response to a number of stimuli, one of which is believed to be aberrant protein targeting to the plasma surface. In this study, the presence of human cytochrome P450 enzyme CYP4Z1 on the outer surface of the plasma membrane of MCF-7 breast cancer cells is demonstrated by immunofluorescence. Moreover, anti-CYP4Z1 aAbs could clearly be detected in breast cancer patient sera by both immunoprecipitation and western blot analysis, while weak or no signals were obtained when testing controls. In an indirect CYP4Z1 ELISA, reactive aAb titers were significantly higher in breast cancer patient samples than in controls. High-resolution epitope mapping of five breast cancer patient sera revealed strong recognition of an epitope that is located on the surface of the enzyme. Cytochrome P450 enzymes (CYPs or P450s) are a superfamily of hemecontaining monooxygenases that are widely distributed in every domain of life. Many of the 57 human CYP enzymes are involved in the bioconversion of xenobiotics, which includes hepatic drug metabolism and the bioactivation of chemical carcinogens, but some family members have their predominant role in the biosynthesis of physiologically important compounds such as steroids and fatty acids. However, these two groups are not strictly separated from each other and overlaps exist; for instance, some CYPs involved in steroid hydroxylation are less specific than once thought and can also oxidize foreign compounds with related structures. Human CYPs are membranebound enzymes that are either located on the cytoplasmic side of the endoplasmic reticulum or on the matrix side of the inner mitochondrial membrane; for their activity they typically depend on specific electron transfer proteins that are co-localized with them. 1 Interestingly, several studies have demonstrated that some microsomal CYPs (such as CYP1A2, CYP2D6 and CYP2E1) as well as cytochrome P450 reductase are transported through secretory vesicles from the endoplasmic reticulum to the outer surface of rodent or human hepatocytes where they face the extracellular space and are catalytically active. Such plasma membrane-localized CYPs are probably one cause for the emergence of anti-CYP aAbs found in patients with a number of liver diseases as well as in patients with some endocrine or autoimmune disorders; for instance, CYP21A2 is the major adrenal cortex autoantigen in idiopathic Addison’s disease, 2 and CYP2D6 is the molecular target of anti-LKM1 (liver kidney microsomal type 1) Abs, which are detectable in type II autoimmune hepatitis and in some patients with HCV hepatitis. 3, 4 All CYPs for which plasma membrane localization has been shown are also known antigens. However, anti-CYP aAbs in cancer patients have not been reported to date, despite the fact that a number of human CYPs (including members of the families CYP1-4 as well as CYP19A1) are known to be overexpressed in various types of malignancies, such as breast, colorectal, lung or ovarian cancer. One of these enzymes is CYP4Z1, which is selectively expressed in mammary tissue and almost undetectable in other healthy human tissues, but displays a strong overexpression in breast cancer and ovarian cancer cells. 5, 6 We have previously shown that CYP4Z1 catalyzes the monohydroxylation of lauric and myristic acid, although it is not known whether this activity is its major physiological function. 7 In this study, we investigated by indirect immunofluorescence microscopy whether CYP4Z1 might also be detected on the plasma membrane of cells of the human epithelial breast cancer …