G-quadruplex or G4 DNA is a stable, four-stranded DNA structure formed from guanine-rich regions. Based on the hypothesis that G4 DNA participated in the regulation of transcription, we analyzed G4 DNA in 5 kb 5' flanking regions of 2892 chicken RefSeq genes with annotated transcription start sites (TSS). In total, 4769 distinct putative G4 DNA motifs (G4M) were identified in 1880 (65%) genes. The pattern of distribution of the G4M showed a gradient along the 5' flanking regions; from -5 to -4 kb, to -1 kb to the TSS, the frequency (number of G4M per kilobase) increased significantly from 0.192 to 0.768, and 62.56% of the G4M in the 1 kb upstream regions were located in the region -400 to the TSS, where a core promoter is always present. Thus, 38.24% of the analyzed genes contained at least one G4M in the 400 bp upstream region. Our findings support the hypothesis that G4M are involved in gene transcription. (c) 2007 Elsevier Inc. All rights reserved.