Glutamate has been shown to be a neurotransmitter in the central nervous system of vertebrates, and it has been hypothesized that glutamate is functional as a neurotransmitter in the spinal cord dorsal horn. A monoclonal antibody to fixative-modified glutamate was used in this study to examine the light microscopic and ultrastructural profiles of glutamate-like immunoreactivity in the superficial dorsal horn of the rat spinal cord. Glutamate-like immunoreactivity was observed in neurons, fibers, and terminals of both laminae I and II. Marginal zone immunoreactive neurons ranged from 10 to 30 micron in diameter and received many nonimmunoreactive somatic synapses. In substantia gelatinosa, immunoreactive neurons were observed in both inner and outer layers, ranged 5 to 10 micron in diameter, and received few nonimmunoreactive somatic synapses. Glutamate-like immunoreactive dendrites were observed in both laminae and were contacted primarily by nonimmunoreactive synaptic terminals that generally contained small clear vesicles. Both myelinated and unmyelinated immunoreactive axons were observed in Lissauer's tract. Immunoreactive terminals contained small (40 nm) clear vesicles and generally formed simple synaptic contacts with nonimmunoreactive dendrites in laminae I and II. The results of this study corroborate the importance of glutamate as a neurotransmitter in spinal sensory mechanisms.