There is growing evidence that mammalian AMP-activated protein kinase (AMPK) plays a role in protecting cells from stresses that cause ATP depletion by switching off ATP-consuming biosynthetic pathways. The active form of AMPK from rat liver exists as a heterotrimeric complex and we have previously shown that the catalytic subunit is structurally and functionally related to the SNF1 protein kinase from Saccharomyces cerevisiae. Here we describe the isolation and characterization of the two other polypeptides, termed AMPKbeta and AMPKgamma, that together with the catalytic subunit (AMPKalpha) form the active kinase complex in mammalian liver. Sequence analysis of cDNA clones encoding these subunits reveals that they are related to yeast proteins that interact with SNF1, providing further evidence that the regulation and function of AMPK and SNF1 have been conserved throughout evolution. The amino acid sequence of the beta subunit is most closely related to SIP2 (35% identity), while the amino acid sequence of the gamma subunit is 35% identical with SNF4. We show that both AMPKbeta and AMPKgamma mRNA and protein are expressed widely in rat tissues. We show that AMPKbeta interacts with both AMPKalpha and AMPKgamma in vitro, whereas AMPKalpha does not interact with AMPKgamma under the same conditions. These results suggest that AMPKbeta mediates the association of the heterotrimeric AMPK complex in vitro, and will facilitate future studies aimed at investigating the regulation of AMPK in vivo.