AMP-activated protein kinase (AMPK) is a fuel sensor in glucose, lipid, and cholesterol metabolism. Using RT-PCR and Western blot, AMPK subunits mRNAs (alpha1/2, beta1/2, and gamma1/2) and proteins (alpha1/2 and beta1/2) can be found in the hen preovulatory follicles and precisely in both granulosa and theca cells. These preovulatory follicles are organized in a hierarchy according to their size (F5/6 to F1). The smallest number (F1) corresponds to the largest size and the latest mature stage. Phosphorylation of AMPKalpha on Thr172 and of acetyl-CoA carboxylase on Ser79 are higher in F4 and F3 than in F1 granulosa cells. However, they are not affected in F4-F1 theca cells. Treatment with 1 mM 5-amino-imidazole-4-carboxyamide-1-beta-D-ribofuranoside (AICAR), an activator of AMPK, dose dependently increased phosphorylation of AMPKalpha on Thr172 in primary F3/4 and F1 granulosa cells. In the absence of FSH, AICAR treatment increased progesterone, P450 side chain cleavage and steroidogenic acute regulatory (StAR) production in both F3/4 and F1 granulosa cells. However, in the presence of FSH, AICAR treatment for 36 h increased progesterone secretion, StAR protein levels and reduced extracellular signal-regulated kinase (ERK)1/2 phosphorylation in F3/4 granulosa cells. Opposite data were observed in F1 granulosa cells. Adenovirus-mediated expression of dominant-negative AMPK totally restored the effects of AICAR on FSH-induced progesterone secretion, StAR protein production, and ERK1/2 phosphorylation in F3/4 and F1 granulosa cells. Using a specific inhibitor of ERK1/2 (U0126), we also showed that this kinase is a negative regulator of the FSH-induced progesterone secretion in F3/4 and F1 granulosa cells, suggesting that AICAR-mediated AMPK activation modifies FSH-induced progesterone secretion differently through the ERK1/2 signaling pathway in hen F3/4 and F1 granulosa cells.