Wound healing involves multiple cell-signaling pathways, including those regulating cell-extracellular matrix adhesion. Previous work demonstrated arachidonate oxidation to leukotriene-B4 by 5-lipoxygenase signals fibroblast spreading on fibronectin, whereas cyclooxygenase-2 catalyzed prostaglandin-E2 formation facilitates subsequent cell migration. Here we investigate arachidonate metabolite signaling in wound closure of perturbed NIH-3T3 fibroblast monolayers. We find during initial stages of wound closure (0-120 minutes), all wound-margin cells spread into the wound gap perpendicular to the wound-long axis. At regular intervals, between 120-300 minutes, some cells elongate to project across the wound and meet cells from the opposite margin, forming distinct cell bridges spanning the wound that act as foci for later wound-directed cell migration and resulting closure. 5-lipoxygenase inhibition by AA861 demonstrated a required leukotriene-B4 signal for initial marginal cell spreading and bridge formation, both of which must precede wound-directed cell migration. 5-lipoxygenase inhibition effects are reversible by exogenous leukotriene-B4. Conversely, cyclooxygenase inhibition by indomethacin reduced directed migration into the wound but enhanced early cell spreading and bridge formation. Exogenous prostaglandin-E2 reversed this effect and increased cell migration into the wound. The differential effects of arachidonic acid metabolites produced by lipoxygenase and cyclooxygenase were further confirmed with NIH 3T3 fibroblast cell lines constitutively over and under-expressing the 5-lipoxygenase and cyclooxygenase-2 enzymes. These data suggest that two competing oxidative enzymes in arachidonate metabolism, lipoxygenase and cyclooxygenase, differentially regulate sequential aspects of fibroblast wound closure in vitro.