Evoked release of acetylcholine and subsequent cell-cell adhesive contacts between growth cones and acetylcholine sensing neurons were observed using cultured neurons dissociated from the diagonal band of Broca of the rat. Stimulation to the soma of the diagonal band of Broca neurons evoked release of acetylcholine from the growth cones. The release of acetylcholine was monitored using whole-cell patch-clamp recording from acetylcholine receptor-rich superior cervical ganglion neuron positioned on the growth cone as a sensor of acetylcholine release. By measuring changes in fluorescence from the growth cone using Ca2(+)-sensitive dye while voltage-clamping the superior cervical ganglion neuron, transient intracellular Ca2+ concentration increase and acetylcholine release from growth cone were recorded simultaneously. Video-enhanced differential interference contrast imaging of the growth cones demonstrated tether formation between the growth cone and superior cervical ganglion cell soma when the superior cervical ganglion cell soma was moved away from the growth cone after acetylcholine release, suggesting formation of adhesive contacts between the growth cone and the sensor neuron. Adhesive contacts between growth cones and sensor neurons were also detected when a high K+ solution or alpha-latrotoxin was applied to the growth cone. Adhesions were also observed between growth cones and latex beads, when growth cones were exposed to high K+ solution. The properties of the adhesive contacts at the growth cone were studied by optically manipulating a latex bead attached to the growth cone surface. These results suggest that growth cones exhibit cell-cell adhesion after neurotransmitter release.