In addition to the ubiquitously expressed form of nonmuscle myosin II heavy chain-B (MHC-B), the existence of a neuron-specific MHC-B isoform, which contains a 30-nucleotide inserted sequence near the ATP binding region, has been reported (Takahashi, M., Kawamoto, S., and Adelstein, R. S.(1992) J. Biol. Chem. 267, 17864-17871). In this study, the genomic location of the neuron-specific inserted 30-nucleotide sequence found in the cDNA is determined to be a single cassette type exon, N30, in the human nonmuscle MHC-B gene. Inclusion or exclusion of exon N30 is cell type-specific, with inclusion being restricted to neuronal cells and being regulated during cell differentiation. Expression of a minigene construct that contains the alternative exon N30 along with the flanking introns and exons was studied in human neuronal retinoblastoma Y79 cells. Inclusion of the N30 exon in the mRNA from the transfected minigene occurs in differentiated Y79 cells that have been treated with butyrate but not in the undifferentiated Y79 cells and non-neuronal cell lines. Systematic deletion and mutation analysis of the minigene construct established that neuron-specific N30 exon recognition requires a cis-acting RNA sequence located approximately 1.5 kilobases downstream of the N30 exon.