Chromatin analyses have identified two DNase I hypersensitive sites (HS1 and HS2) at comparable distances (-130 bp and -2.3 kb) from the transcription start site of the human and mouse alpha2I collagen gene. Whereas the DNA region encompassing HS1 has been extensively characterized using protein binding and functional assays, nothing is yet known about the contribution of the HS2 sequence to alpha2I collagen gene transcription. Here we report that the HS2 sequence of the human alpha2I collagen (COL1A2) gene is a binding site for a transcriptional repressor in fibroblasts. DNase I footprinting identified a single site of nuclease protection around HS2, which corresponds to a sequence potentially capable of forming a 13 bp long hairpin structure with a 4 bp interruption in the middle. Gel mobility shift assays revealed that two GATA consensus sequences embedded within the hairpin are specifically bound by GATA-4 in fibroblasts. They also showed that formation of the HS2 protein complex requires the integrity of the whole hairpin sequence. Transient transfections of luciferase reporter gene constructs in fibroblasts correlated the HS2 element with transcriptional repression of the -2.3 kb promoter sequence. This last observation was further corroborated by showing that forced overexpression of GATA-4 in cultured fibroblasts leads to decreased transcription from the co-transfected -2.3 kb promoter/reporter construct, as well as reduced expression of the endogenous collagen gene. Finally, a chromatin immunoprecipitation assay documented GATA-4 ability to bind to the HS2 element in vivo. These results are therefore the first to implicate GATA-4 in regulating constitutive COL1A2 gene expression in fibroblasts.