We describe a method for visualizing the relative spatial distribution of autophosphorylated Ca2+/calmodulin-dependent protein kinase II (CaMKII) in neuronal subcompartments within hippocampal slices. The method employs a monoclonal antibody recognizing only autophosphorylated CaMKII, and an affinity-purified polyclonal rabbit antisera recognizing only nonphosphorylated CaMK II (Patton et al. (1993) Mol. Biol. Cell, 4: 159-172). 50 microns sections cut from fixed 500 microns hippocampal slices are double-labeled with these antibodies bound by secondary antibodies coupled to fluorescein and Cy3, respectively. The distribution of the two antigens in identical optical sections is recorded by dual channel confocal laser scanning microscopy (CLSM). The digital images are analyzed with the program MacPhase to determine the relative levels of staining with antibodies to phosphokinase and antibodies to nonphosphokinase in subcellular domains of neurons. Comparison of data from paired control and experimental slices reveals the spatial distributions of changes in levels of autophosphorylated CaMKII produced by pharmacological treatments. We are able to detect and spatially resolve differences in levels of autophosphorylation of CaMK II between slices subjected to Ca2+ depletion (low autophosphorylation) and slices treated with a phosphatase inhibitor (high autophosphorylation).