The plant hormone abscisic acid (ABA) regulates important developmental and stress responses. Recent data show that ABA activates phosphorylation events, but whether dephosphorylation events are post-translationally regulated by ABA or whether these are constitutive remains unknown. Slow anion channels in the plasma membrane of guard cells have been proposed to play an important role during ABA-induced stomatal closing. Anion channels are deactivated by removal of cytosolic ATP. However, when guard cells were treated with ABA and depleted of ATP, anion currents remained active. Subsequent removal of extracellular ABA caused deactivation of currents. Deactivation of currents was reversed by reintroduction of cytosolic MgATP. These data show that anion channels are regulated by ABA even in the absence of cytosolic ATP required for kinase-induced phosphorylation events and that anion channel activity is maintained by ABA under conditions that favor dephosphorylation-induced deactivation. Furthermore, channel activation proceeded at high ATP concentrations with nanomolar cytosolic Ca2+ showing a Ca2+-independent final step in anion channel activation.