We have isolated cDNA clones encoding three separate forms of human myelin basic protein (MBP), 21.5, 18.5, and 17.2 kDa, and have determined the nucleotide sequence of each. The three forms share a common sequence but differ by the inclusion of a 26-residue amino acid sequence near the N terminus of the 21.5-kDa protein or by the absence of an 11-residue amino acid sequence near the C terminus of the 17.2-kDa protein. The sequences either added to or deleted from the major 18.5-kDa MBP correspond exactly to exons 2 and 5 of the mouse MBP gene, suggesting that the human and mouse genes have similar exon structures. We have also identified the 21.5-kDa human MBP on immunoblots using antisera raised to a peptide encoded by the mouse exon 2 sequence. Southern blotting studies of human genomic DNA reveal a simple pattern consistent with a single human MBP gene. Thus, the three MBP mRNAs are likely to arise from alternative splicing of a primary human MBP transcript. Conservation of the 26 amino acid mouse exon 2 sequence in human MBP suggests an important role for this sequence in myelination.