Cholinergic neurons isolated from the buccal ganglia of Helisoma were plated into cell culture with a variety of defined target cells to study the specificity of synaptogenesis. Motoneuron B19 selectively formed chemical connections with single dissociated muscle fibers derived from its appropriate target, the supralateral radular tensor (SLT) muscle. B19 did not form such connections with novel neuronal targets. In contrast to neuron B19, cholinergic neuron B5 nonselectively formed chemical connections with novel muscle and neuronal targets. Target cells were micromanipulated into contact with presynaptic neurons to examine the latent period until the onset of functional synaptic transmission. Neuron B5 formed chemical connections within the first minutes of contact with ACh-sensitive neurons and muscle while B19 required sustained periods of muscle-specific contact to induce the acquisition of a functional excitation-secretion coupling mechanism. These different latent periods from the onset of target contact suggest that neuron B5 acquires presynaptic secretory function before target contact, while B19 must receive a specific signal(s) from its appropriate target to induce the transformation of its terminal into a secretory state.