The distribution of glycine immunoreactivity in the lamprey (Lampetra fluviatilis and Ichthyomyzon unicuspis) spinal cord was studied at the light and electron microscopic levels by use of postembedding techniques and antibodies against glutaraldehyde-conjugated glycine. To determine if glycine may be co-stored with other amino acid transmitters, the levels of glycine immunolabeling in identified GABAergic and glutamatergic synapses were examined. The most intense glycine labeling occurred in axon profiles of different diameter distributed throughout the ventral and lateral columns, with the highest density in the areas bordering the lateral cell column. Intermediate levels of glycine labeling were present in certain interneurons in the lateral cell column and in stretch receptors (edge cells) at the lateral margin of the spinal cord. Most other cell bodies, including glutamatergic dorsal cells, were virtually unlabeled. Examination of adjacent sections incubated with GABA antiserum revealed that many of the glycine-containing cells and fibers also contained high levels of GABA. At the ultrastructural level, the glycine immunolabeling was accumulated in two morphologically distinct types of terminal, one of which co-contained GABA. The terminals which exhibited glycine, but not GABA immunoreactivity, contained flattened synaptic vesicles and formed symmetrical synaptic specializations. The terminals that exhibited both GABA and glycine labeling contained pleomorphic synaptic vesicles and had either symmetrical or asymmetrical synaptic specializations. In both cases the glycine labeling was accumulated over the synaptic vesicles. Examination of identified glutamatergic axons in glycine-labeled sections did not provide any evidence for an accumulation of glycine in the synaptic vesicles or other structures of these exons. The present study provide the first morphological description of the localization of glycine in the lamprey spinal cord. The results confirm previous physiological and pharmacological studies, which have implicated glycine as a major fast inhibitory transmitter in the interneuronal network for locomotion, and in a proportion of stretch receptor neurons. The data also show that a significant proportion of the GABAergic synapses, but not the glutamatergic synapses, may utilize glycine as co-transmitter.