Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an evolutionarily conserved glycolytic enzyme, is constitutively expressed in most cell types yet is induced to high levels during the development of fast twitch muscle fibers. To analyze the organization and regulation of the chicken GAPDH gene, we first constructed a nearly full-length GAPDH cDNA clone (pGAD-28). pGAD-28 was used in the current study to screen a genomic library, and several overlapping clones were selected. The GAPDH coding region was detected within a 4.65-kilobase Xho I/EcoRI genomic fragment that was completely sequenced by using the M13 cloning vector system. A small portion of pGAD-28 was used as a primer to extend a 33-nucleotide sequence from the 5' end of GAPDH mRNA. The canonical promoter "TATA" region was found 22 base pairs from the 5' end of the mRNA. The 5' end of the GAPDH gene is extraordinarily G+C-rich (80%) and contains two inverted sequences with a 9-base-pair homology found at -58 (G-G-G-G-C-G-G-G-C) and -93 (G-C-C-C-G-C-C-C-C) nucleotides from the transcription start site. Sequencing also revealed the location of 11 introns within the transcribed portion of the GAPDH gene. The placement of at least 3 of the introns corresponds to the boundaries of protein domains within prokaryotic and eukaryotic GAPDHs that were previously detected by x-ray crystallography. This concordance suggests that introns may have participated in the construction of the earliest GAPDH gene.