The transduction of many cellular stimuli results in oscillations in the intracellular concentration of calcium ions (Ca2+). Although information is thought to be encoded in the frequency of such oscillations, no frequency decoder has been identified. Rapid superfusion of immobilized Ca2+- and calmodulin-dependent protein kinase II (CaM kinase II) in vitro showed that the enzyme can decode the frequency of Ca2+ spikes into distinct amounts of kinase activity. The frequency response of CaM kinase II was modulated by several factors, including the amplitude and duration of individual spikes as well as the subunit composition and previous state of activation of the kinase. These features should provide specificity in the activation of this multifunctional enzyme by distinct cellular stimuli and may underlie its pivotal role in activity-dependent forms of synaptic plasticity.