We have carried out a large-scale identification and characterization of human genes that activate the NF-κB and MARK signaling pathways. We constructed full-length cDNA libraries using the oligo-capping method and prepared an arrayed cDNA pool consisting of 150 000 cDNAs randomly isolated from the libraries. For analysis of the NF-κB signaling pathway, we introduced each of the cDNAs into human embryonic kidney 293 cells and examined whether it activated the transcription of a luciferase reporter gene driven by a promoter containing the consensus NF-κB binding sites. In total, we identified 299 cDNAs that activate the NF-κB pathway, and we classified them into 83 genes, including 30 characterized activator genes of the NF-κB pathway, 28 genes whose involvement in the NF-κB pathways have not been characterized and 25 novel genes. We then carried out a similar analysis for the identification of genes that activate the MARK pathway, utilizing the same cDNA resource. We assayed 145 000 cDNAs and identified 57 genes that activate the MARK pathway. Interestingly, 27 genes were overlapping between the NF-κB and the MAPK pathways, which may indicate that these genes play cross-talking roles between these two pathways.