The sarco/endoplasmic reticulum Ca(2+)-transport ATPase (SERCA2) pre-mRNA is alternatively processed in a tissue-specific manner. At its 3' end, two 5' splice donor sites compete for the same 3' acceptor splice site (3'A). While the upstream 5' donor splice site (5'D1) is used in muscle cells giving rise to the class 1 mRNA, the downstream one (5'D2) is exclusively used in neuronal cells generating the class 4 mRNA. Using a neuroblastoma cell line and a minigene containing the 3' end of the SERCA2 gene, we have investigated the regulation of the neuronal-type of splicing. We have shown that a strong 3'A is required for splicing because exchanging it for a weaker one abolishes splicing. A second region spanning the entire exon 25 downstream of the 3'A is also necessary for the repression of the muscle-specific splicing in neuronal cells. In addition the tissue-specific (muscle/neuron) selection of the appropriate 5' donor splice site seems to be determined by at least two distinct but adjacent negative cis-active elements located in the last 237 nt of the optional exon 24. The upstream negative element controls the neuronal splicing while the downstream one represses the muscle-specific splicing in neuronal cells. It is suggested that the cis-active elements in the gene transcript are the target of trans-acting factors that are responsible for the repression of neuronal- or muscle-specific splicing in a tissue-specific manner.