The AMP‐activated protein kinase (AMPK) is a heterotrimeric complex composed of a catalytic subunit (a) and two regulatory subunits (β and γ). Two isoforms of the catalytic subunit (αl and (α2) have been identified. We show here that the αl‐ and α2‐containing complexes contribute approximately equally to total AMPK activity in rat liver. Furthermore, expression of al or a2 with β and Y in mammalian cells demonstrates that both complexes have equal specific activity measured with the SAMS peptide. Using variant peptides, however, we show that al and a2 exhibit slightly different substrate preferences, which suggest that the two isoforms could play different physiological roles within the cell.