Incubation of either purified rat forebrain Ca2+/calmodulin-dependent protein kinase II (CaMKII) or the purified recombinant, baculovirus-expressed mouse alpha subunit of CaMKII (Brickey, D. A., Colbran, R. J., Fong, Y.-L., and Soderling, T. R. (1990) Biochem. Biophys. Res. Commun. 173, 578-584) with EGTA, magnesium acetate, and [gamma-32P]ATP resulted in the incorporation of up to 1.0 mol of 32PO4/mol of subunit within 60 min at 30 degrees C; both serine and threonine residues became autophosphorylated. The Vmax for this basal autophosphorylation was 0.051 +/- 0.005 mol of 32PO4/mol of subunit/min, and the Km(ATP) was 145 +/- 8 microM. Vmax and Km(ATP) values for Ca2+/calmodulin-dependent autophosphorylation were determined to be 1.3 +/- 0.4 mol/mol/min and 19 +/- 2 microM, respectively. Basal autophosphorylation resulted in inactivation of Ca2+/calmodulin-dependent kinase activity toward exogenous peptide substrate; there was no measurable increase in Ca(2+)-independent CaMKII activity. Inactivation was not observed following preincubation with non-hydrolyzable ATP analogs in place of ATP. CaMKII that had been inactivated by basal autophosphorylation could be fully re-activated by incubation with the purified catalytic subunit of protein phosphatase 2A. Following basal autophosphorylation, the calmodulin-binding ability of CaMKII was also reduced, presumably accounting for the observed inactivation. Both the inactivation and the decrease in calmodulin-binding that resulted from basal autophosphorylation were abrogated by mutation of threonine 306 to alanine, but not by mutation of threonine 305 to alanine. Furthermore, mutation of threonine 306, but not threonine 305, to alanine reduced the extent of basal autophosphorylation at threonine residues. Thus, basal autophosphorylation at threonine 306 blocks calmodulin binding, resulting in inactivation of CaMKII.