Previously we identified a novel 6.5 kb mRNA transcribed from the Duchenne muscular dystrophy (DMD) gene. This mRNA differs in coding content and tissue distribution from the known muscle type and brain type 14 kb DMD mRNAs which code for dystrophin. The novel transcript shares with dystrophin most of the sequence coding for the cysteine-rich and C-terminal domains. Here we used cDNA cloning to identify the divergence point between the common region and the sequence unique to the novel mRNA at the 5' end of the sequence encoding the cysteine-rich domain of dystrophin. This unique sequence containing the translation initiation site is located in a new exon in the intron between exons 62 and 63 of the dystrophin gene. Using probes containing RNA sequences specific to the novel mRNA, we investigated the expression of this mRNA in various tissues and cell types. The study reveals that this mRNA is the main DMD gene product detectable in a variety of nonmuscle tissues including brain cells. The amount of this mRNA in some tissues is comparable to the amount of dystrophin mRNA in the muscle. The expression of the 6.5 kb mRNA is down-regulated during differentiation of myogenic cells; it is present in small amounts in proliferating myoblasts but is undetected in differentiated muscle cultures depleted of mononucleated cells.