Although the sequencing of the human genome has been completed, the number and identity of genes contained within it remains to be fully determined. We used LongSAGE to analyze 660,357 human transcripts from human brain mRNA and identified expression of 17,409 known genes and >15,000 different transcripts that were not annotated in genome databases. Analysis of a subset of these unannotated transcripts suggests that 85% were differentially expressed in various tissue types and that fewer than 20% would have been detected by ab initio gene predictions. These studies suggest that the human genome contains on the order of twice as many transcribed regions as are currently annotated and that experimental approaches will be required to fully elucidate the novel genes corresponding to these transcripts.