Bacterially expressed heterotrimeric (alpha1, beta1, and gamma1) wild-type, catalytically inactive, and constitutively active forms of AMP-activated protein kinase (AMPK) were used to study phosphorylation by an upstream AMPK kinase preparation. Here, we report the identification of two new phosphorylation sites in the alpha-subunit, viz. Thr258 and Ser485 (Ser491 in the alpha2-subunit) by mass spectrometry, in addition to the previously characterized Thr172 site. Also, autophosphorylation sites in the beta1-subunit were identified as Ser96, Ser101, and Ser108. Mutagenesis of Thr172, Thr258, and Ser485 to acidic residues to mimic phosphorylation in the recombinant proteins indicated that Thr172 was involved in AMPK activation, whereas Thr258 and Ser485 were not. Transfection of the non-phosphorylatable S485A and T258A mutants in CCL13 cells subjected to stresses known to activate AMPK either by increasing the AMP:ATP ratio (slow lysis) or without changing adenine nucleotide concentrations (hyperosmolarity) resulted in no significant differences in AMPK activation. All three sites within the alpha-subunit were phosphorylated in vivo, as seen in AMPK immunoprecipitated from anoxic rat liver. In transfected CCL13 cells, the level of Ser485 phosphorylation did not change upon AMPK activation. The newly identified phosphorylation sites could play a subtle role in the regulation of AMPK, e.g. in subcellular localization or substrate recognition.