Natural antisense transcription is now recognized as being a common occurrence in the mammalian transcriptome, and a means by which gene expression can be regulated. In release 1.0, RNAdb contained a dataset of putative antisense ncRNAs identified from cDNA and EST databases for human and mouse using a computational pipeline. Coinciding with release 2.0, we have recently re-developed this pipeline, and experimentally validated a subset of its predictions (Engstrom et al., 2006). We will continue to use the improved pipeline in regular updates of the antisense ncRNA dataset.