Detection of a repellent factor, such as a semaphorin (Sema), causes localized collapse of the growth cone and directs the neurite away from the repellent. Growth cone collapse results from concomitant cytoskeletal rearrangements and detachment of adhesion sites from the extracellular matrix, via mostly unknown signaling mechanisms. In cultures of dorsal root ganglion neurons, we found that Sema3A treatment stimulates the synthesis of the eicosanoid, 12(S)-hydroxyeicosatetraenoic acid (HETE), whereas Sema3A-induced growth cone collapse is prevented when 12(S)-HETE synthesis is blocked with an inhibitor of 12/15-lipoxygenase (LO). Exogenously applied product of 12/15-LO, 12(S)-HETE, mimics Sema3A-induced collapse. As observed by interference reflection and confocal microscopy, 12(S)-HETE causes the loss of growth cone adhesion sites. The adhesion site effect seems partially independent of the actin cytoskeleton because growth cones treated with Sema3A and 12/15-LO inhibitor remain spread despite actin cytoskeleton loss. These studies demonstrate that 12/15-LO activity is a necessary step in Sema3A collapse signaling in growth cones and suggest a mechanism for its action.