Recent transcriptomic studies revealed that extensive proportions of genomes are transcribed, despite the limited fraction of protein-coding gene loci in the whole genome. Most transcripts are considered to be 'cryptic' output of the genome because of the lack of functional evidence; however, recent progress in molecular analyses has revealed that some of these transcripts at least have functional significance. This review article examines evidence of the functional significance of endogenous cis-antisense transcripts, which are the transcriptional output from the opposite strand of annotated genes. These transcripts are one of the most common types of transcripts that do not correspond to any protein-coding loci. Historical molecular studies revealed the existence of antisense transcripts associated with dozens of gene loci, whereas more recent genome-wide studies have shown that many genes have an antisense counterpart thus stimulating investigations into the functional significance of endogenous antisense transcripts. Here, we summarize the recent progress in the genome-wide characterization of the antisense transcriptome, and discuss the biological mechanisms that underlie the regulatory machinery of eukaryotic gene expression with respect to the potential roles of endogenous cis-antisense transcripts.