Oocyte maturation is promoted by the sequential actions of several kinases, of which MPF (a histone H1 kinase) and MAP kinase (a myelin basic protein (MBP) kinase) are known to play pivotal roles. However, other kinases responsible for inducing oocyte maturation have yet to be characterized. To identify these kinases, we examined phosphorylation activities toward 44 exogenous substrate proteins during oocyte maturation in goldfish and Xenopus. We found that 4 substrates in goldfish and 6 in Xenopus were phosphorylated and their phosphorylation states changed during oocyte maturation. Among them, only 3 substrates (histone H1, MBP and pepsin) were common to both species. Precipitation of cdc2 showed that, like histone H1, pepsin was also phosphorylated by cdc2 (MPF). These results suggest that different kinase cascades are involved in goldfish and Xenopus oocyte maturation, although MPF and MAP kinases are common to both species. We also found novel phosphorylation activities that precede the activation of MPF and MAP kinases using deoxyribonuclease I, casein, pepsin and protamine as exogenous substrates.