The C-terminal domain (CTD) of the largest subunit of RNA polymerase II plays critical roles in the initiation, elongation and processing of primary transcripts. These activities are at least partially regulated by the phosphorylation of the CTD by three cyclin-dependent protein kinases (CDKs), namely CDK7, CDK8 and CDK9. In this study, we systematically compared the phosphorylation of different recombinant CTD substrates by recombinant CDK7/CycH/MAT1, CDK8/CycC and CDK9/CycT1 kinases. We showed that CDK7, CDK8 and CDK9 produce different patterns of phosphorylation of the CTD. CDK7/CycH/MAT1 generates mostly hyperphosphorylated full-length and truncated CTD peptides, while CDK8/CycC and CDK9/CycT1 generate predominantly hypophosphorylated peptides. Total activity towards different parts of the CTD also differs between the three kinases; however, these differences did not correlate with their ability to hyperphosphorylate the substrates. The last 10 repeats of the CTD can act as a suppressor of the activity of the kinases in the context of longer peptides. Our results indicate that the three kinases possess different biochemical properties that could reflect their actions in vivo.