In an attempt to identify glutamatergic neurons, the cerebral cortex and thalamus of the rat were examined immunohistochemically by using a monoclonal antibody against phosphate-activated glutaminase (PAG), a major synthetic enzyme of transmitter glutamate in the central nervous system. In both the neocortex and mesocortex, pyramidal cells in layers V and VI showed intense PAG-like immunoreactivity (PAG-LI), whereas neuronal cell bodies in layers I-IV showed weak PAG-LI. At the deep border of layer VI, neurons with horizontally elongated cell bodies showed PAG-LI. In the pyriform and entorhinal cortices, neurons with intense to moderate PAG-LI were seen in layer II as well as in the deeper layers. In the hippocampal formation, pyramidal cells in CA1, CA2, and CA3 and polymorphic cells in CA4 showed PAG-LI; PAG-LI was most intense in pyramidal cells of CA3. Fine granules with weak PAG-LI were also seen on and/or within the cell bodies of granule cells in the dentate gyrus. In the thalamus, neurons with PAG-LI were distributed in all nuclei, although regional differences were observed in the distribution pattern of neurons with PAG-LI and in the intensity of PAG-LI in individual neurons. The largest neurons in each thalamic nucleus showed intense PAG-LI; these were considered to be projection neurons. In addition to perikaryal labeling, many fine, PAG-like immunoreactive granules were distributed in the neuropil of both the cerebral cortex and thalamic nuclei. Some of these fine granules with PAG-LI in the neuropil were assumed to represent fiber terminals with PAG-LI, because the distribution pattern of the deposits in the primary somatosensory and primary visual cortices resembled that of thalamocortical fiber terminals. Glutamate is rather ubiquitous in the mammalian central nervous system, and it is still debatable whether the monoclonal antibody to PAG from brain mitochondria can distinguish transmitter-related glutaminase from the other metabolism-related ones. In the present study, however, large neurons in the thalamic nuclei, as well as pyramidal neurons in the cerebral cortex, showed PAG-LI most intensely, supporting the assumption that projection neurons of the cerebral cortex and thalamus are primarily glutamatergic.