"Perineuronal nets" (PNs) ensheath a subtype of inhibitory neurons in the mammalian neocortex. In the light of the proposal that PNs consist of glial processes, we have analyzed the relationship between intracellularly injected glial cells and PNs in the rat neocortex. Glial cells were injected iontophoretically with Lucifer Yellow in lightly fixed tissue slices and PNs were visualized with the lectin from Vicia villosa. Using confocal laser scanning microscopy, glial processes and PNs were identified as distinct structures. Lectin labeling was consistently associated with the extracellular space interposed between LY-labeling was consistently associated with the extracellular space interposed between LY-labeled astrocyte processes and neurons. Of the different types of glial cells injected, only the densely-ramifying protoplasmic astrocytes extended processes which could be traced to contact PNs. These protoplasmic astrocytes also sent out processes to adjacent neurons not ensheathed by PNs, and to capillaries. The present data strongly suggests that PNs do not consist of glial processes but rather support the idea that PNs represent specialized extracellular material interposed between the surface of some inhibitory interneurons and astrocytic processes.