A mouse brain beta-spectrin of cDNA was identified within a lambda Gt11 expression library using an antibody which specifically binds with the 235 kDa spectrin beta-subunit. Restriction mapping and DNA sequencing analyses of the brain cDNA revealed that this clone contained 1185 bp of sequence, of which a 999 bp single open reading frame encoding 333 amino acids was determined. The deduced amino acid sequence exhibited homology with beta-spectrins, demonstrating the characteristic 106 amino acid repeating unit. The homology between our mouse brain sequence and human RBC beta-spectrin was approximately 56% beginning at the beta 15 repeat unit and extending to the C-terminus of sequence elucidated for human RBC sequence. An additional 62 amino acids were found at the C-terminus of the 235 kDa brain beta-spectrin subunit not seen in the human RBC sequence. The approximately 1.2 Kb brain spectrin cDNA insert hybridized with a single 9 Kb mRNA transcript in various adult mouse tissues, with the most abundant hybridization demonstrated in RNA isolated from brain tissue. This mRNA was found to be present at high levels in heart tissue and at lower levels in spleen and skeletal muscle tissue. The 9 Kb mRNA was different in content and in size to mRNAs which hybridized with a cDNA encoding the mouse erythroid beta-spectrin subunit, demonstrating that the brain spectrin cDNA is a distinct gene product and represents the first known sequence of a nonerythroid beta-spectrin subunit.