The dodecameric holoenzyme of calcium-calmodulin-dependent protein kinase II (CaMKII) responds to high-frequency Ca(2+) pulses to become Ca(2+) independent. A simple coincidence-detector model for Ca(2+)-frequency dependency assumes noncooperative activation of kinase domains. We show that activation of CaMKII by Ca(2+)-calmodulin is cooperative, with a Hill coefficient of approximately 3.0, implying sequential kinase-domain activation beyond dimeric units. We present data for a model in which cooperative activation includes the intersubunit 'capture' of regulatory segments. Such a capture interaction is seen in a crystal structure that shows extensive contacts between the regulatory segment of one kinase and the catalytic domain of another. These interactions are mimicked by a natural inhibitor of CaMKII. Our results show that a simple coincidence-detection model cannot be operative and point to the importance of kinetic dissection of the frequency-response mechanism in future experiments.