Ovulated rat oocytes are activated spontaneously soon after recovery from the oviducts. To investigate the kinetics and mechanism of rat oocyte spontaneous activation (OSA), we investigated the effect of aging in oviducts, hyaluronidase treatment, and extracellular and intracellular calcium, and examined the activity of CaMKII and the effect of its inhibitor on OSA. Oocyte aging in oviducts and hyaluronidase did not affect OSA. However, OSA was significantly decreased in calcium-free medium and in calcium-containing medium containing L-type calcium channel blocker and IP3R inhibitor. Moreover, significantly lower OSA was shown with an inhibitor of CaMKII. There was a significant increase of CaMKII activity at 30 min after oocyte recovery and constitutively active CaMKII was located near the meiotic spindle in freshly recovered oocytes. Therefore, CaMKII is one of the upstream signals to activate rat oocytes spontaneously after recovery and rat oocytes respond very sensitively to extracellular calcium. Crown copyright (c) 2007 Published by Elsevier Inc. All rights reserved.