We hypothesized that circulating polymorphonuclear granulocytes (PMNs), vascular endothelial cells (ECs), and perivascular mast cells (MCs) may initiate and sustain the inflammatory response through the generation of the superoxide anion (O₂·^-) by PMNs primed by inflammatory stimuli, which in turn evoked the overexpression of adhesion molecules from ECs and release of histamine by MCs. To pin-point the role of carbon monoxide (CO) in curbing vascular inflammation, we studied the effect of a water-soluble CO-releasing molecule [tricarbonylchloro-glycinate-ruthenium (II); CORM-3] on an experimental model of vascular inflammation. The model consists of coincubating formyl-methionyl peptide (fMLP) -primed human PMNs with rat ECs or with rat MCs. The effects of CORM-3 were evaluated by measuring the generation of O₂·^- and the expression of CD11b in fMLP-primed PMNs; the expression of ICAM-1 and CD203c in ECs and MCs, respectively; and the release of histamine from MCs. Our results show that the chemotactic peptide fMLP primes PMNs to generate O₂·^- and overexpress CD11b, both events being central to the inflammatory process, while CORM-3 significantly decreases these events (IC₅₀=1.66 µM for O₂·^- production; 1.20 µM for CD11b expression in human PMNs). The experiments also show that fMLP-primed PMNs increase the CD54 expression by coincubated ECs, and the expression of CD203c and the release of histamine by coincubated MCs. Once again, CORM-3 abolishes these events (IC₅₀=6.78 µM for CD54 expression in ECs; 1.18 µM for CD203 expression; 1.15 µM for histamine release in MCs). Thus, CORM-3 exerts a powerful anti-inflammatory action by down-regulating the oxidative burst in PMNs, the overexpression of adhesion molecules in PMNs and ECs, the release of histamine, and the overexpression of an activation marker by MCs.—Masini, E., Vannacci, A., Failli, P., Mastroianni, R., Giannini, L., Vinci, M. C., Uliva, C., Motterlini, R., Mannaioni, P. F. A carbon monoxide-releasing molecule (CORM-3) abrogates polymorphonuclear granulocyte-induced activation of endothelial cells and mast cells.