Putative aspartergic and glutamatergic sensory neurons in the rat were identified by autoradiography and immunocytochemistry respectively. Approximately 3% of large L4 dorsal root ganglion neurons (diameter 18–52 μm) accumulated radiolabelled aspartate, whereas all satellite glia had high affinity for the amino acid. Glutamate-immunofluorescent (Glu-FITC) dorsal root ganglia neurons comprised 38.3% at S1, 35.6% at L2 33.9% at C5 and 28.8% at T6. Numbers of immunoreactive neurons were higher with the more sensitive peroxidase-anti-peroxidase (Glu-PAP) method; and the cell counts totalled 42% (S1), 41.2% (L4), 35% (C5) and 34.6% (T6). The trigeminal ganglion (TG) contained 24% Glu-FITC and 32.3% Glu-PAP positive cells. The majority of glutamate-immunoreactive sensory neurons were small, ranging from 10–35 μm with median diameters of 17.5μm (C5), 21μm (S1), 24.2μm (TG) and 28.5 μm (L2). It is evident therefore, that a subgroup of class B cells are glutamatergic. Glutamate immunoreactivity in the spinal cord was similar in all segments and was localized in the superficial lamina and substantia gelatinosa of the dorsal horn. Stained interneurons were located among the immunoreactive fibres. The dorsolateral funiculus contained dense plexus of immunoreactive fibres which increased in prominence after intraperitoneal injection of L-glutamate, but penetration of exogenous glutamate into the grey matter was limited. Instead, the meninges and basal layers of the spinal blood vessels were intensely immunoreactive. The studies describe the subtypes of acidic amino acidergic neurons and relates the immunohistochemistry to a functional subclass.