The 3' end of Acetylcholinesterase (AChE) pre-mRNA is processed by a complex mechanism of alternative splicing. Three different transcripts are generated and called R, H and T according respectively to the intron (intron 4') or exons (5 or 6) retained in the mature RNA. The relative expression of the specific transcripts depends on cell type, developmental stage or pathophysiological conditions. The aim of our study was to identify sequences involved in AChE pre-mRNA splicing choices. For this purpose, we constructed a minigene in which the constitutive exons were fused and followed by the entire alternative domain without 3' UTR. We transfected the wild-type or minigene mutated in the alternative domain in muscle or COS-7 cells and identified the splicing products by RPA, RT-PCR and sedimentation coefficients of the enzymatic molecular forms. We find that the alternative splicing domain contains most of the necessary signals to control splicing choices in skeletal muscle cells with the coding sequences of the domain having little effect on the splicing outcome. A branch point at an unusual location 278 nt from the 3' acceptor site of exon 6 is characterized. We further identify several regulatory sequences in the non-coding sequence of exon 5 that regulate the splicing pattern. Sequences that control the splice to exon 5 and those which influence intron 4' retention or splicing to exon 6 appear to be distinct.