PSD-95, a prominent protein component of the postsynaptic density (PSD) fraction from rat forebrain, has been localized by light microscopy to dendrites of hippocampal neurons (Cho et al., 1992) and to the presynaptic plexus of cerebellar basket cells (Kistner et al., 1993). Here we extend these studies to show that an affinity-purified antibody to PSD-95 labels the dendrites of most neurons in the forebrain and of a subset of neurons in the cerebellum. To confirm that PSD-95 is associated with the PSD at forebrain synapses and to clarify whether it is also associated with the presynaptic membrane, we employed immunogold electron microscopy of forebrain synaptosomes. Gold-labeled antibodies to PSD-95 labeled postsynaptic densities in both intact and lysed forebrain synaptosomes but did not label presynaptic terminals or the presynaptic membrane. The asymmetric distribution of PSD-95 at synapses contrasts with that of its homologs, disks-large and ZO-1, which are arranged symmetrically at septate and tight junctions, respectively.