Non-coding RNAs are involved in epigenetic processes, playing a role in the regulation of gene expression at the transcriptional and post-transcriptional levels. A particular group of ncRNA are natural antisense transcripts (NATs); these are transcribed in the opposite direction to protein coding transcripts and are widespread in eukaryotes. Their abundance, evidence of phylogenetic conservation and an increasing number of wellcharacterized examples of antisense-mediated gene regulation are indicative of essential biological roles of NATs. There is evidence to suggest that they interfere with their corresponding sense transcript to elicit concordant and discordant regulation. The main mechanisms involved include transcriptional interference as well as dsRNA formation. Sense-antisense hybrid formation can trigger RNA interference, RNA editing or protein kinase R. However, the exact molecular mechanisms elicited by NATs in the context of these regulatory roles are currently poorly understood. Several examples confirm that ectopic expression of antisense transcripts trigger epigenetic silencing of the related sense transcript. Genomic approaches suggest that the antisense transcriptome carries a broader biological significance which goes beyond the physiological regulation of the directly related sense transcripts. Because NATs show evidence of conservation we speculate that they played a role in evolution, with early eukaryotes gaining selective advantage through the regulatory effects. With the surge of genome and transcriptome sequencing projects, there is promise of a more comprehensive understanding of the biological role of NATs and the regulatory mechanisms involved.