Recently we discovered that Fzr1/Cdh1 activity is essential to maintain prophase I stage arrest in fully-grown mouse oocytes by mediating degradation of cyclin B1. Fzr1 is an activator on the Anaphase-Promoting Complex (APC), a multisubunit E3 ubiquitin ligase responsible for polyubiquitinating substrates, such as cyclin B1, that in so being are earmarked for degradation through the 26S proteasome. Cyclin B1 is the regulatory subunit of Maturation-Promoting Factor, forming a heterodimer with CDK1, whose kinase activity requires cyclin B1 binding and is essential for dissolution of the nuclear envelope and condensation of chromatin associated with entry into M-phase. APCFZR1 activity would therefore maintain prophase I arrest by degrading cyclin B1 and in so doing prevent a rise in CDK1 kinase activity. However, with respect to oocytes we currently do not understand how Fzr1 activity is fine tuned, such that cyclin B1 is maintained at sufficient levels to allow oocytes to resume meiosis but not at too high a level to resume meiosis prematurely. Here we describe how intracellular partitioning would explain how this regulation is achieved. Using an oocyte cDNA library, generated from prophase I oocytes in Swiss Quackenbush mice, we found only one splice variant, Fzr1α/Cdh1α, of the single-copy mammalian fzr1 gene, in contrast to various splice variants we found in other tissues. This gene product contained a predicted nuclear localization signal. By immunofluorescence we confirmed this nuclear location, both in isolated oocytes from mice and in oocytes contained in antral follicles from ovarian sections. In fact all the degradation machinery appeared enhanced in the nucleus, as observed by immunofluorescence of APC6, an essential APC subunit and PSMD11, an essential 26S proteasome subunit. In contrast, cyclin B1 was predominantly cytoplasmic. In all systems studied it has been demonstrated that cyclin B1 shuttles between the cytoplasm and nucleus, with nuclear localization occurring just before nuclear envelope breakdown. We reasoned therefore that the nuclear localization of the APCFZR1 and 26S proteasome would aid in maintaining low nuclear levels of cyclin B1. To test this idea further we used two cyclin B mutants, coupled to GPF, which differed in their intracellular location. F146A-cyclin B1, which accumulates in the nucleus due to a single point mutation in the nuclear export signal, and 5A-cyclin B1 in which 5 S/T residues, phosphorylated on cyclin B1 entry into the nucleus, are alanine substituted. We found that nuclear accumulation of cyclin B1 was necessary in order for it to promote meiotic resumption because over-expression of F146A-cyclin B1 accelerated entry into meiosis, whereas 5A-cyclin B1 did not despite the same level of expression. However, in oocytes maintained in prophase I arrested through addition of milrinone, rates of F146A-cyclin B1 degradation were 5 fold (6% vs 32%) that of 5A-cyclin B1. We conclude that in oocytes, an increase in the nuclear-cytoplasmic ratio of cyclin B1 is an essential step in meiotic resumption. During prophase I the constant shuttling of cyclin B1 between nucleus and cytoplasm presents the possibility of precocious meiotic entry, but we argue that this is physiologically prevented by nuclear APCFZR1 activity. Research supported by the NHMRC (Grant 569202) to KTJ. (platform)