An assay employing patterned laminin substrata was used to screen for compounds that disrupt neurite guidance. One molecule, pertussis toxin, caused neurites to wander from patterns that normally guided them, yet had no significant effect on rates of neurite outgrowth. Wandering was greatest on patterns requiring frequent guidance (e.g., laminin stripes with periodic gaps). Surprisingly, the B oligomer of pertussis toxin, which lacks the subunit that inactivates G proteins, was equipotent at disrupting neurite guidance. Pertussis toxin probably acts by binding cell surface carbohydrates, since neurites lacking complex-type N-linked oligosaccharides were insensitive to the effects of the toxin. The B oligomer also blocked growth cone collapse induced by a brain membrane-derived factor; such factors are thought to act as repulsive guidance cues in vivo. That a single reagent can inhibit neuronal responses to both attractive and repulsive guidance cues suggests that such cues may share signaling pathways.