The human brain contains four isoforms of myelin basic protein (MBP), previously identified by cDNA cloning. We have now isolated and characterized genomic clones encoding the human MBP gene. The gene is 45 kb in extent and consists of seven exons. Alternative splicing of the primary MBP transcript can account for all four human MBP isoforms. The intron-exon boundaries of the gene have also been determined, and all conform to the known consensus splice sequences. These sequences, however, do not explain the alternative splicing pattern found in human brain. Transcription of the human MBP gene begins at a single site within the MBP promoter, and all four MBP isoforms are transcribed from this same site. The promoter region does not contain any known sequence elements, but does have a 12-bp sequence also found in the JC virus 98-bp tandem repeat. A relative gradient of MBP transcription is found from caudal to rostral within the developing human brain, which parallels the known sequence of myelination found in these areas. RNase protection of brain RNA demonstrates more of the 21.5-kD and 20.5-kD MBP mRNAs in neonatal brain than in the adult frontal cortex, which suggests that alternative splicing of the primary MBP transcript is also regulated temporally during myelin development. These data show that regulation of myelination is complex, involving regional cellular interactions and trans activation of transcription, as well as modulation of alternative splicing. Comparison of the human and mouse data also suggests that alternative splicing plays an important role in myelin biogenesis.