Cytochrome P450 (CYP) epoxygenases metabolize endogenous polyunsaturated fatty acids to their corresponding epoxides, generating bioactive lipid mediators. The latter play an important role in vascular homeostasis, angiogenesis, and inflammation. However extra-endothelial sources of EETs or their functional importance on endothelial cells is less investigated. EETs could be detected in freshly isolated human monocytes with an increase in the concentration during the differentiation to macrophages. Mass spectrometric analysis from microsomal fraction identified CYP2S1 as a source of the epoxides of arachidonic, linoleic and eicosapentaenoic acid. Analyzing the expression of CYP2S1 in classical inflammatory (M1) or alternatively activated phenotypes (M2) revealed that LPS and IFN-γ increased the expression of CYP2S1 mRNA whereas IL-4 and IL-13 had no effect on expression. The immunohistochemical analysis of inflamed human tonsils showed a high level of CYP2S1 expression that is colocalized with CD68. In addition to its epoxygenase function Cyp2s1 can act as hydroxylase on PGG2 and PGH2, immediate precursors of prostaglandin (PG) E2 converting them to 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT). CYP inhibition and siRNA-mediated downregulation of CYP2S1 increased macrophage phagocytosis and that the latter effect correlated with decreased 12-HHT formation. Although no Cyp2s1 protein was detected in aortae from wild-type mice it was expressed in aortae and macrophage foam cells from ApoE-/- mice. Consistent with these observations CYP2S1 was colocalised with the monocyte marker CD68 in human atherosclerotic lesions. In summary, CYP2S1 generates 12-HHT and is a novel regulator of macrophage function that is expressed in classical inflammatory macrophages, and can be found in atherosclerotic plaques.