SummaryFor the full activation of cyclin‐dependent kinases (CDKs), not only cyclin binding but also phosphorylation of a threonine (Thr) residue within the T‐loop is required. This phosphorylation is catalyzed by CDK‐activating kinases (CAKs). In Arabidopsis three D‐type CDK genes (CDKD;1–CDKD;3) encode vertebrate‐type CAK orthologues, of which CDKD;2 exhibits high phosphorylation activity towards the carboxy‐terminal domain (CTD) of the largest subunit of RNA polymerase II. Here, we show that CDKD;2 forms a stable complex with cyclin H and is downregulated by the phosphorylation of the ATP‐binding site by WEE1 kinase. A knockout mutant of CDKD;3, which has a higher CDK kinase activity, displayed no defect in plant development. Instead, another type of CAK – CDKF;1 – exhibited significant activity towards CDKA;1 in Arabidopsis root protoplasts, and the activity was dependent on the T‐loop phosphorylation of CDKF;1. We propose that two distinct types of CAK, namely CDKF;1 and CDKD;2, play a major role in CDK and CTD phosphorylation, respectively, in Arabidopsis.