Electron microscopic immunocytochemistry provides an important tool to determine the ultrastructural distribution of various molecules in both normal and pathologic tissues. However, the specific immunostaining may be obscured by artifactual immunoreaction product, misleading the investigator. Previous observations show that shortening the incubation period with the primary antibody from the generally used 12-24 hours to 1 hour substantially reduces the artifactual immunostaining. We now extend this finding by the demonstration of artifact-free ultrastructural localization of the Ca2/calmodulindependent cyclic nucleotide phosphodiesterase (CaM-dependent PDE) immunoreactivity in brain.Anesthetized rats were perfused transcardially with phosphate-buffered saline followed by a fixative containing paraformaldehyde (4%) and glutaraldehyde (0.25%) in PBS. The brains were removed, and 40μm sections were cut with a vibratome. The sections were processed for immunocytochemistry as described by Ludvig et al. Both non-immune rabbit serum and specific CaM-dependent PDE antibodies were used. In both experiments incubations were at one hour and overnight. The immunostained sections were processed for electron microscopic examination.