We have cloned a cDNA encoding a novel B regulatory subunit of protein phosphatase 2A (PP2A) from a Xenopus oocyte cDNA library. The novel B subunit, termed B beta', shows the strongest overall sequence similarity to, but a distinct N-terminal sequence from, the beta isoform of the human/rat B subunit. When expressed ectopically in Xenopus oocytes, the B beta' isoform can augment the endogenous PP2A activity and inhibit oocyte maturation induced by progesterone. These results suggest that the B beta' isoform can form a complex with other PP2A subunits to make a trimeric PP2A holoenzyme in Xenopus oocytes and may negatively control the initiation of oocyte maturation.