Type IV collagen is present ubiquitously in basement membranes. A bifunctional promoter regulates the expression of the α1/α2 genes, and the α3/α4 and the α5/α6 genes are also considered to be regulated by putative bifunctional promoters. Unlike the other type IV collagen chains, the α5(IV) and α6(IV) chains do not always co-localize and are present in distinct basement membranes. To address such dichotomy in the α5(IV) and α6(IV) gene regulation, we cloned a mouse genomic DNA fragment containing the promoter region between the two transcription start sites of these genes and we then placed this putative promoter sequence between the chloramphenicol acetyltransferase and Luciferase reporter genes, so that these genes would be transcribed in opposite directions in this unique construct. Glomerular endothelial cells and mesangial cells generate the kidney glomerular basement membrane, which always contains the α5(IV) chain but not the α6(IV) chain. In contrast, the basement membranes of Bowman's capsule and distal tubuli (produced by the tubular epithelial cells) contain the α6(IV) chain. We demonstrate that, in response to TGF-β (transforming growth factor β), epidermal growth factor, vascular endothelial growth factor and platelet-derived growth factor, expression from the α5(IV) gene is significantly enhanced in the glomerular endothelial cells and mesangial cells, but not expression from the α6(IV) gene. In contrast, the expression from the α6(IV) gene, and not that from the α5(IV) gene, was significantly enhanced in response to growth factors in the tubular epithelial cells. Our results demonstrate that the proximal bifunctional promoter regulates the expression of the α5(IV) and α6(IV) genes in a cell-specific manner and offers the first demonstration of the promoter plasticity in growth factor regulation of type IV collagen genes in different tissues of the body.