A cDNA clone for the α subunit of mouse brain Ca²⁺/CaM-dependent protein kinase II (CaM-kinase II) was transcribed in vitro and translated in a rabbit reticulocyte lysate system. Inclusion of [³⁵S]methionine in the translation system yielded a single ³⁵S-polypeptide of about 50 kDa. When the translation system was assayed for CaM-kinase II activity, there was a 5-10-fold enrichment of kinase activity which was totally dependent on Ca²⁺/calmodulin (CaM). Both the 50-kDa 35S-polypeptide and the Ca²⁺/CaM-dependent protein kinase activity were quantitatively immunoprecipitated by rat brain CaM-kinase II antibody. When the translated wild-type kinase was subjected to autophosphorylation conditions in the presence of Ca²⁺, CaM, Mg²⁺, and ATP, the Ca²⁺-independent activity (assayed in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid) increased from 5.8 ± 0.7 to 26.5 ± 2.1% of total activity (assayed in the presence of Ca²⁺/CaM). These properties confirm the identity of the kinase translated in vitro as CaM-kinase II. The role of Thr-286 autophosphorylation in formation of the Ca²⁺-independent activity was investigated by site-directed mutation of Thr-286 to Ala (Ala-286 kinase) and to Asp (Asp-286 kinase). The Ala-286 kinase was completely dependent on Ca²⁺/CaM for activity prior and subsequent to autophosphorylation. The Asp-286 kinase exhibited 21.9 ± 0.8% Ca²⁺-independent activity, and this was not increased by autophosphorylation. These results establish that introduction of negative charge(s) at residue 286, either by autophosphorylation of Thr or by mutation to Asp, is sufficient and necessary to generate the partially Ca²⁺-independent form of CaM-kinase II.