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Recent work describing the transcriptional output of the human genome points to the existence of a significant number of non-coding RNA transcripts coming from intronic regions, with a fraction of these being oriented antisense relative to the protein-coding mRNA of the known gene. In this article, we survey the main findings of the large-scale expression analysis projects that led to the identification of antisense intronic messages and which demonstrate their ubiquitous expression in the human genome. We review the current knowledge on long, unspliced, intronic antisense transcripts, a new class of non-coding RNAs, recently described by our group to be correlated with the degree of tumor differentiation in prostate cancer, which we postulate is involved in the fine tuning of gene expression in eukaryotes. Possible mechanisms of antisense intronic transcript biogenesis and function in gene expression regulation are discussed, as is their involvement in human diseases. While there is still no conclusive evidence demonstrating a functional role for these long, intronic antisense messages, the far-reaching implications of their existence for the mechanisms regulating gene expression certainly warrant further experimentation.