The purpose of this study was to examine the effects of an activator of AMPK (5-aminoimidazole-4-carboxamide 1-beta-D-ribofuranoside (AICAR)) on bovine oocyte nuclear maturation in vitro. After 7 hr of culture, AICAR (1 mM) significantly increased the percentages of cumulus-enclosed oocytes (CEO) and denuded oocytes (DO) remaining at the germinal vesicle stage. After 22 hr of culture, AICAR significantly reduced the percentage of CEO reaching metaphase II (MII). AICAR at 1.0 mM also increased the inhibitory effect of the adenylate cyclase activator forskolin in CEO; however, at 0.05 mM, AICAR increased the percentage of oocytes at MII after 22 hr of culture compared to forskolin alone. The adenosine kinase inhibitor 5'-aminodeoxyadenosine reversed the effect of AICAR in CEO and DO showing that phosphorylation of AICAR by adenosine kinase is required for its inhibitory activity. GMP, but not AMP, inhibited meiosis in CEO and DO; however, inhibition of guanyl and adenyl nucleotides synthesis did not reverse the effect of AICAR suggesting that the inhibitory effect of AICAR is not due to increased synthesis of these nucleotides. Metformin, another activator of AMPK, also inhibited GVBD in CEO and DO. The alpha-1 isoform of the catalytic subunit of AMPK was detected in oocytes and cumulus cells, and reverse transcription-polymerase chain reaction experiments showed the presence of transcripts for alpha-1, alpha-2, beta-1, and gamma-3 isoforms of the regulatory subunits in cumulus cells and oocytes. These data show that the AMPK activator AICAR is inhibitory to nuclear maturation in bovine oocytes due to activation of AMPK.