AMP-activated protein kinase (AMPK) and Ca2+/calmodulin (CaM)-dependent protein kinase I (CaMKI) are protein kinases that are regulated both by allosteric activation (AMP and Ca2+/CaM, respectively) and by phosphorylation by upstream protein kinases (AMPK kinase (AMPKK) and CaMKI kinase (CaMKIK), respectively). We now report that AMPKK can activate CaMKI and that, conversely, CaMKIK can activate AMPK. CaMKIK is 68-fold more effective at activating CaMKI than AMPK, while AMPKK is 17-fold more effective at activating AMPK than CaMKI. Our results suggest that CaMKIK and AMPKK are distinct enzymes dedicated to their respective kinase targets but with some overlap in their substrate specificities. The availability of alternative substrates for AMPKK and CaMKIK allowed the unequivocal demonstration that AMP and Ca2+/calmodulin promote the activation of AMPK and Ca2+/calmodulin promote the activation of AMPK and CaMKI, respectively, via three independent mechanisms: 1) direct activation of AMPK and CaMKI, 2) activation of AMPKK and CaMKIK, and 3) by binding to AMPK and CaMKI, inducing exposure of their phosphorylation sites. Since AMP and Ca2+/calmodulin each has a triple effect in its respective system, in vivo, the two systems would be expected to be exquisitely sensitive to changes in concentration of their respective activating ligands.