Elevated cytosolic calcium and protein kinase C are well-established mediators of luteolytic actions of prostaglandin F_2alpha (PGF_2alpha). The objectives of this study were to determine 1) if calcium/calmodulin-dependent kinase kinase 2 (CAMKK2) participates in mediating PGF_2alpha actions in developing (Day [d]-4) and mature (d-10) bovine corpus luteum (CL), 2) distal targets of CAMKK2, 3) developmental expression of adenosine monophosphate-activated protein kinase (AMPK), and 4) effects of AMPK activation on progesterone (P4) production. Expression of AMPK increased as the CL matured. Activation of the prostaglandin receptor (FP) induced rapid phosphorylation of AMPK, which was blocked by a CAMKK2 inhibitor. Changes in basal P4 secretion in vitro were determined in response to AMPK activation via metformin (met) or 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) in d-4 and d-10 CL. Production of P4 in d-10 CL decreased with met or AICAR compared to control, similar to activation by PGF_2alpha. Therefore, potential distal targets of AMPK in d-10 CL were examined during induced functional regression via exogenous PGF_2alpha. Serum and luteal P4 decreased at 2 and 4 h after administration of PGF_2alpha. Protein expression of LDLR decreased at 2 and 4 h, while those of ACAT1 and STAR increased 4 h after PGF_2alpha. During induced regression, alterations of cholesterol transport proteins contributed to decreased luteal and serum P4. Therefore, developmental differences in signal transduction associated with FP, specifically CAMKK2 and AMPK, partially contribute to differences in the ability of PGF_2alpha to induce regression in mature, but not developing, bovine CL. Multiple cholesterol transport proteins, including LDLR, were altered by PGF_2alpha and could be potential AMPK targets.