Abstract Background: Previous studies have demonstrated that Cytosolic phospholipase A2a (cPLA2a) is absolutely required for NOX2 NADPH oxidase activation in human and mouse phagocytes. Moreover, upon stimulation, cPLA2a translocates to the plasma membranes of by binding to the assembled oxidase, forming a complex between its C2 domain and the PX domain of the oxidase cytosolic factor, p47phox in human phagocytes. Intravenous administration of an antisense against cPLA2a that significantly inhibited its expression in mouse peritoneal neutrophil and macrophages also inhibited superoxide production, in contrast to cPLA2a knockout mice that showed normal superoxide production. The aim of the present study was to determine whether there is a binding between cPLA2a-C2 domain and p47phox-PX in mouse macrophages, to further support the role of cPLA2a in oxidase regulation also in mouse phagocytes. Methods and Results: A significant binding of mouse GST-p47phox-PX domain fusion protein and cPLA2a in stimulated mouse phagocyte membranes was demonstrated by pull down experiments, although lower than that detected by human p47phox-PX domain. Substituting the amino acids Phe98, Asn99 and Gly100 to Cys98 Ser99 and Thr100 in mouse p47phox-PX domain (that are present in human p47phox-PX domain) caused strong binding that was similar to that detected by the human p47phox-PX domain. Conclusions: the binding between cPLA2a-C2 and p47phox-PX domains exist in mouse macrophages and is not unique to human phagocytes. The binding between the two proteins is lower in the mice probably due to the absence of amino acids Cys98 Ser99 and Thr100 in p47phox-PX domain that facilitate the binding to cPLA2a.