One of the most abundant proteins in postsynaptic densities is identical or very similar to the alpha-subunit of the Ca2+/calmodulin-dependent protein kinase II. Autophosphorylation of this protein in isolated postsynaptic densities was studied under various conditions, following inhibition of endogenous phosphatase activity with microcystin-LR. Phosphorylation accompanied by a shift in the enzyme's electrophoretic mobility was observed upon incubation with Ca2+ and calmodulin at 37 degrees C. Brief incubation with Ca2+ and calmodulin at 0 degrees C resulted in a low level of phosphorylation and no change in mobility. Following this limited Ca(2+)-dependent phosphorylation, however, a high level of phosphorylation could be achieved in the absence of Ca2+, upon incubation at 37 degrees C. Comparison of reverse-phase HPLC phosphopeptide elution profiles obtained following phosphorylation at 37 degrees C, in the presence and absence of Ca2+, as described above, showed differences, suggesting that certain distinct sites may be phosphorylated under each condition. A major phosphopeptide peak, however, with the amino acid sequence Met-Leu-Thr(P)-Ile-Asn-Pro-Ser-Lys was identified under both conditions. This sequence is identical to the predicted sequence containing Thr-253 of the Ca2+/calmodulin-dependent protein kinase II. The results suggest that phosphorylation at Thr-253 requires an initial Ca(2+)-dependent phosphorylation, which may be at a different site, but does not depend on the continued presence of Ca2+ to proceed. The observed mode of regulation of autophosphorylation at Thr-253 appears to be unique to the postsynaptic density-associated enzyme.