Estimates of the number of genes for organisms with a wide range of biological complexity will emerge from genome sequencing projects. This information will be crucial to correlate gene number with biological complexity. Computer-based annotations of the genome sequences of Saccharomyces cerevisiae, Caenorhabditis elegans and Drosophila melanogaster predict approximately 6,000, 19,000 and 13,600 genes, respectively. It is seemingly paradoxical that C. elegans contains 50% more genes than D. melanogaster and that the latter only contains twice as many genes as S. cerevisiae. However these numbers should be considered with caution until gene predictions are verified using experimental approaches. To experimentally verify gene predictions, we PCR amplified predicted open reading frames from a C. elegans cDNA library, cloned them and generated an ORF sequence tag (OST) for each of them. In a random sample (n = 1,200) of the ∼10,000 genes predicted from the entire C. elegans genome but experimentally unverified so far, 90% of these predicted genes could be unambiguously verified by OST sequencing. When added to the list of ∼9,500 genes previously verified by individual laboratories and/or by EST sequencing, our data confirm the existence of at least 19,000 protein-encoding genes in C. elegans. We suggest that ORFeome cloning coupled with OST analysis could be used to verify gene number predictions for other organisms. Furthermore, the demonstration and identification of 10,000 novel genes provides a resource for comparative genomics and gene expression studies. We also discuss our findings in the context of the current debate on the human gene number.