Acetylcholine (ACh) sensitivity and extrajunctional receptor distribution of the rat diaphragm were closely monitored during the early period following denervation. Both contracture in response to 10 microgram/ml of ACh and extrajunctional binding of [125I]alpha-bungarotoxin ([125I]alpha-BTX)) were first detectable 30 h after cutting the phrenic nerve in the thorax. If the nerve were cut more proximally, leaving a 3.5 cm distal nerve stump, the same level of ACh contracture and [125I]alpha-BTX binding did not appear until 40 h after operation. This 10-h delay was far longer than the 3-h delay in transmission failure reportedly dependent on stump length. The earliest detectable extrajunctional [125I]alpha-BTX binding appeared throughout the entire muscle fiber, and was not localized to the endplate region as would be expected if degeneration in the nerve terminal induced new receptors. However, later significant increases in [125I]alpha-BTX binding at the endplate region could have resulted from such degeneration. All these results are consistent with neurotrophic regulation of muscle ACh receptors, working via a mechanism involving axonal transport.