Nerve-muscle synapses form and the postsynaptic membrane begins to differentiate rapidly in vitro. New clusters of AChRs have been located at transmitting synapses within 3 hours after a competent neurite contacted a receptive myotube. Receptors within new subneural clusters are not identical with receptors at adult end-plates: they are metabolized rapidly and their mean channel open time is prolonged. In this sense synapse formation must be considered a drawn out affair. AChE also accumulates at synapses soon after functional contacts are established. The cleft enzyme is apparently regulated differently than receptors in the postsynaptic membrane. Muscle activity is necessary for the early accumulation of AChE, but not for the clustering of receptors. Further studies in vitro should be useful for analysis of the role of muscle activity, of the local neural influence on the postsynaptic membrane, and of the mechanism of the muscle response.