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Mediator is a large complex containing up to 30 subunits that consist of four modules each: head, middle, tail and CDK/Cyclin. Recent studies have shown that CDK8, a subunit of the CDK/Cyclin module, is one of the key subunits of Mediator that mediates its pivotal roles in transcriptional regulation. In addition to CDK8, CDK19 was identified in human Mediator with a great deal of similarity to CDK8 but was conserved only in vertebrates. Previously, we reported that human CDK19 could form the Mediator complexes independent of CDK8. To further investigate thein vivotranscriptional activities of the complexes, we used a luciferase assay in combined with siRNA-mediated knockdown to show that CDK8 and CDK19 possess opposing functions in viral activator VP16-dependent transcriptional regulation. CDK8 supported transcriptional activation, whereas CDK19, however, counteracted it. In this study, we further characterized CDK19. We used microarrays to identify target genes for each CDK, and we selected six genes: two target genes of CDK8, two target genes of CDK19 and two genes that were targets for both. Surprisingly, it turned out that both CDKs bound to all six target genes, regardless of their effects in transcription upon binding, suggesting Mediator as a context-specific transcriptional regulator.