We have previously demonstrated that the a isoform of Ca2+/calmodulin-dependent protein kinase kinase (CaM-KK alpha) is strictly regulated by an autoinhibitory mechanism and activated by the binding of Ca2+/CaM [Tokumitsu, H., Muramatsu, M., Ikura, M., and Kobayashi, R. (2000) J. Biol. Chem. 275, 20090-20095]. In this study, we find that rat brain extract contains Ca2+/CaM-independent CaM-KK activity. This result is consistent with an enhanced Ca2+/CaM-independent activity (60-70% of total activity) observed with the recombinant CaM-KK beta isoform. By using various truncation mutants of CaM-KK beta, we have identified a region of 23 amino acids (residues 129-151) located at the N-terminus of the catalytic domain as an important regulatory element of the autonomous activity. A CaM-KK beta deletion mutant of this domain shows a significant increase of Ca2+/CaM dependency for the CaM-KK activity as well as for the autophosphorylation activity. The activities of CaM-KK alpha and CaM-KK beta chimera, in which autoinhibitory sequences were replaced by each other, were completely dependent on Ca2+/CaM, suggesting that the autoinhibitory regions of CaM-KK alpha and CaM-KK beta are functional. These results establish for the first time that residues 129-151 of CaM-KK beta participate in the release of the autoinhibitory domain from its catalytic core, resulting in generation of autonomous activity.