Maintaining correct DNA methylation patterns entails the addition of methyl groups by DNA methyltransferases and the active removal of methylation from DNA. Removing a methyl group from 5-methylcytosine requires breaking a strong C–C bond, suggesting that demethylation might occur by an alternative mechanism that does not involve severing this bond. Indeed, the discovery of the 5-methylcytosine DNA glycosylase (also known as DNA demethylase) REPRESSOR OF SILENCING 1 (ROS1) by (Gong et al., 2002) revolutionized thinking in this field, as the study of ROS1 revealed a mechanism by which 5-methylcytosine is excised and replaced by the DNA repair machinery. This special issue celebrates the 20th anniversary of the discovery of ROS1 and the remarkable research that followed.