We report evidence for a developmental role of acetylcholine in axon pathfinding in the Drosophila visual system. Acetylcholine was detected on photoreceptor axons during their navigation to target sites in the brain, a time well before the formation of functional synapses. The pattern of photoreceptor axon projections was severely disrupted when acetylcholine synthesis or metabolism was altered or eliminated, or when transgenic alpha-bungarotoxin, a nicotinic acetylcholine receptor antagonist, was expressed in the developing eye or brain. The requirement for acetylcholine signaling exists before photoreceptor neurons form synaptic connections and does not require the function of vesicular acetylcholine transporter protein. That this early effect of acetylcholine is mediated through nonvesicular release is further supported by the observation that transgenic expression of tetanus toxin, a blocker of neurotransmitter release via synaptic vesicles, did not cause similar photoreceptor axon projection defects. These observations support the notion that a form of acetylcholine secretion mediates the behavior of growth cones during axon pathfinding.