Glycosphingolipids (GSLs) and their metabolites play important roles in a variety of biological processes. We have previously reported that lactosylceramide (LacCer), a ubiquitous GSL, stimulates NADPH oxidase-dependent superoxide generation by aortic smooth muscle cells and their consequent proliferation. We postulated that LacCer may upregulate adhesion molecules on human polymorphonuclear leukocytes (hPMNs), perhaps also via NADPH oxidase-dependent reactive oxygen metabolite (ROM) generation. Incubation of hPMNs with LacCer upregulated CD11b/CD18 (Mac-1) and CD11c/CD18, as determined by fluorescence-automated cell sorting. LacCer also stimulated these hPMNs to generate superoxide via NADPH oxidase, as determined by lucigenin-enhanced chemiluminescence. However, the upregulation of Mac-1 by LacCer did not itself appear to be mediated by ROMs, since neither an antioxidant nor an NADPH oxidase inhibitor substantially inhibited the Mac-1 upregulation. However, this Mac-1 upregulation was significantly inhibited by two disparate phospholipase A2 (PLA2) inhibitors. Moreover, LacCer induced arachidonic acid metabolism, which was inhibited by the PLA2 inhibitors, but not by an NADPH oxidase inhibitor. To evaluate the effect of LacCer on hPMN adhesion to endothelium, hPMNs stimulated with LacCer were allowed to adhere to unstimulated human endothelial cell monolayers. LacCer stimulated hPMN adhesion to endothelial cells, which was blocked by anti-CD18 and by the PLA2 inhibitors. We conclude that LacCer stimulates both Mac-1 upregulation and superoxide generation in hPMNs but that ROMs are not the upstream signal for Mac-1 upregulation. This mechanism may well be relevant to acute endothelial injury in inflammation and other pathological conditions.