A second isoform of Ca²⁺/calmodulin-dependent-kinase II inhibitor protein (CaM-KIIN) has been identified using the yeast two-hybrid screen. The 1.8kb message encodes a 78 residue CaM-KIINα that is 65% identical in its putative open-reading frame and 95% identical in its inhibitory domain to the previously characterized CaM-KIINβ. CaM-KIINα exhibits inhibitory properties towards recombinant mouse CaM-kinase IIα indistinguishable from CaM-KIINβ. The 27 amino acid inhibitory peptide (CaM-KIINtide) derived from CaM-KIIN has the ability to inhibit brain CaM-kinase II activity from multiple organisms including rat, Drosophila and goldfish. Northern analysis of various rat tissues indicates that CaM-KIINα is specific to brain whereas CaM-KIINβ message is also present in testis. In situ hybridization shows a general distribution of both isoforms in rat brain with stronger localization of CaM-KIINβ in cerebellum and hindbrain and CaM-KIINα in frontal cortex, hippocampus and inferior colliculus. An antibody that recognizes both isoforms shows a distribution of CaM-KIIN in rat brain that correlates with immunoreactivity of CaM-kinase II. In cultured mature hippocampal neurons, CaM-KIIN is present in cell bodies and dendrites but, unlike CaM-kinase II, does not display punctate staining at synapses. These results suggest a localized function for CaM-KIIN in inhibiting specialized pools of CaM-kinase II.