The lack of a calcium channel agonist (e.g., BayK8644) for CaV2 channels has impeded their investigation. Roscovitine, a potent inhibitor of cyclin-dependent kinases 1, 2, and 5, has recently been reported to slow the deactivation of P/Q-type calcium channels (CaV2.1). We show that roscovitine also slows deactivation (EC50 ~53 µM) of N-type calcium channels (CaV2.2) and investigate gating alterations induced by roscovitine. The onset of slowed deactivation was rapid (~2 s), which contrasts with a slower effect of roscovitine to inhibit N-current (EC50 ~300 µM). Slow deactivation was specific to roscovitine, since it could not be induced by a closely related cyclin-dependent kinase inhibitor, olomoucine (300 µM). Intracellularly applied roscovitine failed to slow deactivation, which implies an extracellular binding site. The roscovitine-induced slow deactivation was accompanied by a slight left shift in the activation-voltage relationship, slower activation at negative potentials, and increased inactivation. Additional data showed that roscovitine preferentially binds to the open channel to slow deactivation. A model where roscovitine reduced a backward rate constant between two open states was able to reproduce the effect of roscovitine on both activation and deactivation. © 2005 by the Biophysical Society.