Mitochondrial ATP synthase gamma-subunit (F(1)gamma) pre-mRNA undergoes alternative splicing in a tissue- or cell type-specific manner. Exon 9 of F(1)gamma pre-mRNA is specifically excluded in heart and skeletal muscle tissues and in acid-stimulated human fibrosarcoma HT1080 cells, rhabdomyosarcoma KYM-1 cells, and mouse myoblast C2C12 cells. Recently, we found a purine-rich exonic splicing enhancer (ESE) element on exon 9 via transgenic mice bearing F(1)gamma mutant minigenes and demonstrated that this ESE functions ubiquitously with exception of muscle tissue (Ichida, M., Hakamata, Y., Hayakawa, M., Ueno E., Ikeda, U., Shimada, K., Hamamoto, T., Kagawa, Y., Endo, H. (2000) J. Biol. Chem. 275, 15992-16001). Here, we identified an exonic negative regulatory element responsible for muscle-specific exclusion of exon 9 using both in vitro and in vivo splicing systems. A supplementation assay with nuclear extracts from HeLa cells and acid-stimulated HT1080 cells was performed for an in vitro reaction of muscle-specific alternative splicing of F(1)gamma minigene and revealed that the splicing reaction between exons 8 and 9 was the key step for regulation of muscle-specific exon exclusion. Polypyrimidine tract in intron 8 requires ESE on exon 9 for constitutive splice site selection. Mutation analyses on the F(1)gammaEx8-9 minigene using a supplementation assay demonstrated that the muscle-specific negative regulatory element is positioned in the middle region of exon 9, immediately downstream from ESE. Detailed mutation analyses identified seven nucleotides (5'-AGUUCCA-3') as a negative regulatory element responsible for muscle-specific exon exclusion. This element was shown to cause exon skipping in in vivo splicing systems using acid-stimulated HT1080 cells after transient transfection of several mutant F(1)gammaEx8-9-10 minigenes. These results demonstrated that the 5'-AGUUCCA-3' immediately downstream from ESE is a muscle-specific exonic splicing silencer (MS-ESS) responsible for exclusion of exon 9 in vivo and in vitro.