Collagen production plays a significant role in tumor development, especially in breast cancer, hepatocarcinomas, and colorectal carcinoma. However, collagen production is decreased during oncogenic transformation of cells in culture. This study demonstrates that methylation of the collagen alpha2(I) gene transcription start site occurs frequently in human cancer cell lines (9 of 10), including breast cancer cell lines (MCF-7 and Hs578T), hepatocellular carcinoma cell lines (SNU387, SNU449, SNU398, and PLC/PRF/5), a fibrosarcoma cell line (HT1080), and colorectal carcinoma cell lines (HCT116, SW480, and SW620). In addition, the collagen gene is more methylated in colorectal cancer tissues compared with normal mucosa. The increased DNA methylation of the collagen gene in cell lines is inversely correlated with collagen mRNA steady-state levels. Most importantly, treatment of fibrosarcoma or breast carcinoma cells with a DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine, resulted in lower methylation and reactivation of the collagen gene in a dose-responsive manner. This is the first demonstration that the collagen alpha2(I) gene is methylated in multiple cancer cell lines correlating with loss of collagen expression and also methylated in primary cancer tissues. These data also suggest that methylation-induced repression of collagen transcription may be a frequent occurrence in cancer.