We created pCOL-KT, a plasmid construct in which the promoter/enhancer of human Pro alpha 1(I) gene is linked to the chloramphenicol acetyl transferase reporter gene. The Pro alpha 1(I) promoter/enhancer in pCOL-KT was methylated in vitro and tested for transcriptional activity by transient expression analysis. Methylation of the construct with bacterial methylases reduced transcriptional activity about 25-fold. Site-specific methylation of eight potential canonical sites of eukaryotic methylation within the promoter greatly reduced transcriptional activity. Chromatin conformation of the transfected pCOL-KT DNA was analyzed by nuclease sensitivity. Although both methylated and unmethylated transfected DNA had increased susceptibility to DNase I compared with the endogenous gene, the methylated transfected DNA showed increased resistance to nuclease when compared with unmethylated transfected DNA, indicating that the methylation of the DNA alters the chromatin conformation. We also tested the ability of a human rhabdomyosarcoma cell line that does not express type I collagen to support transcription from an exogenously added Pro alpha 1(I) promoter/enhancer. The transformed cell line is able to support transcription from the Pro alpha 1(I) promoter/enhancer. Treatment of the transformed cell line with 5-azacytidine, a potent inhibitor of DNA methylation, resulted in transcriptional activation of the Pro alpha 1(I) gene. These findings, along with the extreme methylation sensitivity of the Pro alpha 1(I) promoter and enhancer, suggest that DNA methylation may be an important mechanism of transcriptional inactivation of interstitial collagen genes.