Substantial evidence has indicated that cAMP-dependent protein kinase (protein kinase A or PKA) plays a critical role in maintaining meiotic prophase arrest in vertebrate oocytes. However, PKA activity dynamic and its physiological substrate profile during oocyte maturation remain poorly defined. We have recently developed a novel PKA substrate construct which we employ to monitor PKA activity in live oocytes. We demonstrated here that, during progesterone-induced oocyte maturation, PKA was inactivated within 30 min of the addition of progesterone and thereafter remained inactivated throughout the entire maturation process. However, artificial reactivation of endogenous PKA had differential consequences, depending on the timing of PKA reactivation. Reactivation of endogenous PKA at any time prior to germinal vesicle breakdown (GVBD) inhibited progesterone-induced GVBD. PKA reactivation at GVBD, or thereafter, did not interfere with meiosis I to meiosis II transition, nor did it interfere with metaphase II arrest. These results demonstrate for the first time a PKA-restrictive and a PKA-permissive phase in oocyte maturation.