Both a partial cDNA clone and a complete genomic clone have been isolated for human gfa, the gene encoding the major component of astrocyte intermediate filaments, glial fibrillary acidic protein (GFAP). The nucleotide sequence of the entire coding region and 102 bp of the 5' flanking DNA was determined. The mRNA start site was identified by primer extension and probe protection experiments, and a novel in vitro transcription and translation procedure was then used to establish that the first ATG in the mRNA initiates GFAP synthesis. The predicted amino-terminal sequence for human GFAP differs greatly from that previously deduced for mouse GFAP from its gene sequence, despite otherwise high homology. This discrepancy was resolved by determining that the published mouse genomic sequence has an incorrect additional base. The corrected sequence produces strong homology between human and mouse GFAP in their predicted amino acid sequences, and suggests that human and mouse GFAP initiate at homologous positions. The beginning sequence deduced here for both proteins is matched closely by that previously obtained for porcine GFAP by direct sequencing of its amino-terminal end. This supports the protein initiation sites proposed, and also indicates that GFAP is not processed at its amino-terminal end.