Members of the family of Polo-like kinases are implicated in the regulation of cell cycle progression in all eukaryotes. In Xenopus laevis, only one member of this family, Plx1, has previously been described. Here we report the cloning and characterization of X. laevis Plx2 and Plx3, the likely homologs of mammalian Plk2 (Snk) and Plk3 (Fnk/Prk), respectively. RNA expression studies indicate that all three Xenopus Plks are present in both oocytes and unfertilized eggs. Further analysis by in situ hybridization revealed that Plx1 RNA is ubiquitously expressed in early embryos, but shows more restricted expression at later stages. In contrast, Plx2 and Plx3 expression is highly restricted in both early and late-stage embryos. Using Plx-specific antisera, Plx1 and Plx3 polypeptides could readily be detected on immunoblots of oocyte and egg extracts. Both Plx1 and Plx3 protein levels remained virtually constant during oocyte maturation. However, whereas Plx1 is more active in M phase than in I phase (P. Descombes and E. A. Nigg (1998) EMBO J. 17, 1328-1335), Plx3 protein and activity levels remained constant upon release of meiotic metaphase II-arrested egg extracts into interphase. Finally, microinjection of in vitro-transcribed RNAs for Plx1, Plx2, and Plx3 increased the rate of progesterone-induced oocyte maturation, and concomitantly, all three kinases became activated. Conversely, overexpression of the corresponding catalytically inactive kinases delayed maturation. This suggests that, at least in oocytes, all three kinases may be regulated by similar mechanisms, and they may also share common substrates. However, the strikingly restricted pattern of expression of Plx2 and Plx3 observed in embryos strongly suggests that individual Plk family members perform at least partly distinct functions at later stages of development.