Cetn3 inhibits Mps1 kinase activity in vitro and at centrosomes by blocking activating autophosphorylation and can prevent Mps1 from phosphorylating Cetn2 even when Mps1 is present at 10-fold molar excess. Cetn3 also prevents incorporation of Cetn2 into centrioles, but mimicking phosphorylation of Cetn2 bypasses the inhibitory effects of Cetn3.