Oxidative stress in cells and tissues leads to the formation of an assortment of lipid electrophiles, such as the quantitatively important 4-hydroxy-2- trans-nonenal (HNE). Although this cytotoxic aldehyde is atherogenic the mechanisms involved are unclear. We hypothesize that elevated HNE levels can directly inactivate esterase and lipase activities in macrophages via protein adduction, thus generating a biochemical lesion that accelerates foam cell formation and subsequent atherosclerosis. In the present study we examined the effects of HNE treatment on esterase and lipase activities in human THP1 monocytes/macrophages at various physiological scales (i.e., pure recombinant enzymes, cell lysate, and intact living cells). The hydrolytic activities of bacterial and human carboxylesterase enzymes (pnbCE and CES1, respectively) were inactivated by HNE in vitro in a time- and concentration-dependent manner. In addition, so were the hydrolytic activities of THP1 cell lysates and intact THP1 monocytes and macrophages. A single lysine residue (Lys105) in recombinant CES1 was modified by HNE via a Michael addition reaction, whereas the lone reduced cysteine residue (Cys389) was found unmodified. The lipolytic activity of cell lysates and intact cells was more sensitive to the inhibitory effects of HNE than the esterolytic activity. Moreover, immunoblotting analysis using HNE antibodies confirmed that several cellular proteins were adducted by HNE following treatment of intact THP1 monocytes, albeit at relatively high HNE concentrations (>50 μM). Unexpectedly, in contrast to CES1, the treatment of a recombinant human CES2 with HNE enhanced its enzymatic activity ∼3-fold compared to untreated enzyme. In addition, THP1 monocytes/macrophages can efficiently metabolize HNE, and glutathione conjugation of HNE is responsible for ∼43% of its catabolism. The functional importance of HNE-mediated inactivation of cellular hydrolytic enzymes with respect to atherogenesis remains obscure, although this study has taken a first step toward addressing this important issue by examining the potential of HNE to inhibit this biochemical activity in a human monocyte/macrophage cell line.