Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) is a known activating kinase for AMP-activated protein kinase (AMPK). In vitro, CaMKK phosphorylates Thr(172) in the AMPK subunit more efficiently than CaMKK, with a lower K-m (approximate to 2 m) for AMPK, whereas the CaMKI phosphorylation efficiencies by both CaMKKs are indistinguishable. Here we found that subdomain VIII of CaMKK is involved in the discrimination of AMPK as a native substrate by measuring the activities of various CaMKK/CaMKK chimera mutants. Site-directed mutagenesis analysis revealed that Leu(358) in CaMKK/Ile(322) in CaMKK confer, at least in part, a distinct recognition of AMPK but not of CaMKI.