A monoclonal antibody against phosphate-activated glutaminase was used to identify glutamatergic neuronal components in the intermediolateral nucleus of the thoracic spinal cord of the rat. Under electron microscopy of the intermediolateral nucleus, most glutaminase immunoreactivity was detected in the axoplasm surrounding spherical synaptic vesicles in the presynaptic axon varicosities which formed asymmetric synapses with small dendrites and occasionally with neuronal cell bodies. About 40% of axon varicosities within the intermediolateral nucleus and 49% of the axon varicosities forming asymmetric synaptic contacts showed glutaminase immunoreactivity. Glutaminase immunoreactivity was further seen in mitochondria of neuronal perikarya and dendrites in the intermediolateral nucleus, and occasionally in the cytoplasm of the dendrites and glial processes in the vicinity of glutaminase-immunoreactive axon varicosities. By the combined method of immunocytochemistry and retrograde axonal transport, glutaminase-immunoreactive axons were shown to make direct synaptic contacts with the preganglionic sympathetic neurons, which were retrogradely labeled by injection of horseradish peroxidase conjugated with choleratoxin B subunit into the superior cervical ganglion. The present results indicate that glutaminase-containing axons are the major synaptic inputs to intermediolateral nucleus neurons including preganglionic sympathetic ones, suggesting that glutamate is used as the neurotransmitter to control those neurons in the intermediolateral nucleus.