We studied the splicing and compartmentalization of acetylcholinesterase (AchE) mRNAs during muscle differentiation in the mouse, both in vitro and in vivo. We used the polymerase chain reaction (PCR) to analyse AChE mRNAs in cultures of the myogenic C2 and Sol8 cell lines, and in the developing diaphragm, from embryonic day 14 (E14). We characterized three types of alternatively spliced AChE mRNAs, encoding catalytic subunits that differ by their C-terminal regions (R, H and T). The T transcript is predominant in all cases and represents the only AChE mRNA in the adult muscle. We detected the presence of the minor R and H transcripts in the myogenic cell lines, both as myoblasts and differentiated myotubes, and also in the diaphragm from E14 until birth. At E14 the R transcript represents approximately 1% of AChE mRNA and the level of the H transcript is still lower. By in situ hybridization, we found that the T AChE mRNAs begin to preferentially accumulate at the level of the first neuromuscular contacts in the mouse diaphragm and other muscles as early as E14, e.g. concomitantly with mRNAs encoding the receptor subunits. This suggests that a common control mechanism ensures the synaptic focalization of mRNAs encoding the cholinergic proteins AChE and acetylcholine receptor during muscle development.