Synaptic rearrangements in developing muscle were studied by visualizing individual neuromuscular junctions in the sternomastoid muscle of living neonatal mice as they underwent the transition from multiple to single innervation. Vital staining of ACh receptors (AChRs) with rhodamine-conjugated alpha-bungarotoxin showed that while junctions were still multiply innervated (usually by two motor axons), regions of the postsynaptic membrane within each junction became depleted of receptors. Usually, several small postsynaptic areas lost AChRs in succession. In these areas, AChRs already in the membrane rapidly disappeared compared to a low level of receptor turnover elsewhere in the junction. Moreover, there was no evidence of new AChRs being inserted into these areas. Within each postsynaptic area undergoing AChR depletion, the intensity of receptor staining decreased gradually over 1-2 d. In some junctions, it appeared that AChRs were migrating away from areas being depleted of receptors. The depletion of AChRs from some sites in combination with the spreading apart of the entire receptor-rich area due to muscle fiber growth accounts for the transformation from plaque-like to branched receptor distributions at developing neuromuscular junctions. Vital staining of presynaptic motor nerve terminals at junctions whose postsynaptic AChRs were also stained showed that motor nerve terminals were lost from the same areas that were depleted of receptors postsynaptically. Postsynaptic areas began to be depleted of AChRs before there was any obvious loss of membrane or intracellular staining in the overlying nerve terminal. Only when a single innervating axon remained at a junction did loss of motor nerve terminals and underlying AChRs largely cease. That former synaptic areas could at later times be identified as uninnervated regions within a junction indicates that synapse elimination during development leaves an indelible mark on synaptic structure. These observations suggest that the withdrawal of a motor axon from a neuromuscular junction occurs as a consequence of the stepwise elimination of all of its synapses with that muscle fiber. These results also suggest that an important aspect of synaptic competition leading to axon withdrawal is the precocious loss of AChRs beneath the nerve terminals of the axon that will be eliminated. A similar early loss of AChRs beneath one axon's synapses has been shown to occur during synapse elimination in reinnervated adult muscle (Rich and Lichtman, 1989a).(ABSTRACT TRUNCATED AT 400 WORDS)