Abstract: In Saccharomyces cerevisiae cells, B-type cyclin-dependent kinases (CDKs) target two origin recognition complex (ORC) subunits, Orc2 and Orc6, to inhibit helicase loading. We show that helicase loading by ORC is inhibited by two distinct CDK-dependent mechanisms. Independent of phosphorylation, binding of CDK to an "RXL" cyclin-binding motif in Orc6 sterically reduces the initial recruitment of the Cdt1/Mcm2-7 complex to ORC. CDK phosphorylation of Orc2 and Orc6 inhibits the same step in helicase loading. This phosphorylation of Orc6 is stimulated by the RXL motif and mediates the bulk of the phosphorylation-dependent inhibition of helicase loading. Direct binding experiments show that CDK phosphorylation specifically blocks one of the two Cdt1-binding sites on Orc6. Consistent with the inactivation of one Cdt1-binding site preventing helicase loading, CDK phosphorylation of ORC causes a twofold reduction of initial Cdt1/Mcm2-7 recruitment but results in nearly complete inhibition of Mcm2-7 loading. Intriguingly, in addition to being a target of both CDK inhibitory mechanisms, the Orc6 RXL/cyclin-binding motif plays a positive role in the initial recruitment of Cdt1/Mcm2-7 to the origin, suggesting that this motif is critical for the switch between active and inhibited ORC function at the G1-to-S-phase transition.