Abstract The location of neuromuscular junctions which form in vitro between regenerating adult mouse muscle fibres and sections of embryonic mouse spinal cord was examined. The position of the original motor end-plates on the explanted muscle fibres was determined by using either rhodamine-labelled α-bungarotoxin (RαBT) binding to the acetylcholine receptors, or by stains to demonstrate acetylcholinesterase (AChE) also located at the end-plate. In this culture system, the explanted muscle fibres degenerate and regenerate to form new myotubes which develop cross-striations and contractions. The location of the newly-formed neuromuscular junctions in these mature cultures was then demonstrated using RαBT-binding to acetylcholine receptors, silver impregnation and cholinesterase techniques. Less than half the new neuromuscular junctions were at the original end-plate areas indicating that, at least in this system, junctions can form at sites other than those of the original end-plate.