Type IV collagen, a major component of the basement membrane (BM), is composed of six genetically distinct alpha(IV) chains, alpha1(IV) to alpha6(IV). Their genes are paired on three different chromosomes in a head-to-head arrangement. The alpha5(IV) gene (COL4A5) and the alpha6(IV) gene (COL4A6) are on chromosome Xq22 and are regulated by a bidirectional promoter. Loss of the alpha5(IV)/alpha6(IV) chains in epithelial BM occur in the early stage of cancer invasion. However, the regulatory mechanism of the specific loss of the alpha5(IV)/alpha6(IV) chains during cancer cell invasion is still undetermined. In the present study, we examined the expression of the alpha5(IV)/alpha6(IV) chains and the methylation profiles of the bidirectional promoter region of COL4A5/COL4A6 in colon cancer cell lines and colorectal tumor tissues. The expression of the alpha5(IV)/alpha6(IV) chains was down-regulated in colorectal cancer, and the loss of expression of the alpha5(IV)/alpha6(IV) chains was associated with the hypermethylation of their promoter region. In conclusion, the hypermethylation of the bidirectional promoter region of COL4A5/COL4A6 is one of the events that is responsible for the loss of expression of the alpha5(IV)/alpha6(IV) chains and the remodeling of the epithelial BM during cancer cell invasion.