Pathfinding by growing nerve processes in the developing nervous system depends on the turning response of the growing tip, the growth cone, to extracellular guidance cues. There is evidence in vivo and in cell culture that some growth cones exhibit chemotropic behaviour, but the identity of endogenous chemoattractants remains elusive. Neurotransmitters appear early in the developing embryo and may have morphogenic roles in development. In cell culture a number of neurotransmitters were found to induce growth inhibition or retraction of neurites. Here we report positive turning responses of the nerve growth cone in a defined extracellular gradient of the neurotransmitter acetylcholine (ACh). The growth cone response depends on the activation of neuronal nicotinic ACh receptors, requires the presence of extracellular Ca2+, and appears to be mediated by Ca(2+)-calmodulin-dependent protein kinase II. Fluorescence imaging of cytosolic Ca2+ concentration ([Ca2+]i) at the growth cone showed a small but significant evaluation of [Ca2+]i within minutes of the onset of ACh application and before the turning of the growth cone. These findings suggest that neurotransmitters may serve as specific chemoattractants for growth cone guidance and that cytosolic Ca2+ may act as a second messenger in the cytoplasm of the growth cone to initiate the turning response.