A quantitative immunocytochemical method was used to study the regional levels of glutamate in two types of lamprey (Ichtyomyzon unicuspis) axon, which both activate excitatory amino acid receptors, but which when active exhibit different firing patterns. Giant reticulospinal axons fire in brief bursts, while dorsal column axons, mainly belonging to cutaneous afferents, show a sustained firing at high frequency. In both types of axon, clusters of synaptic vesicles showed a strong accumulation of glutamate immunogold labeling, and the density of gold particles correlated strictly with the packing density of synaptic vesicles. The most densely packed vesicle areas had a particle density corresponding to a concentration of fixed glutamate of about 30 mM in coprocessed glutamate conjugates, suggesting an intravesicular glutamate concentration of at least 60 mM. The level of labeling in axoplasmic matrix was considerably lower, but differed significantly between the two types of axon. Dorsal column axons showed a particle density in axoplasmic matrix that was approximately four times higher than that in giant reticulospinal axons. The mitochondrial glutamate labeling was also significantly stronger in the dorsal column axons. In addition, the number of mitochondrial profiles surrounding vesicle clusters was about four times higher in dorsal column synapses than in reticulospinal synapses. Antisera to aspartate, GABA, glutamine, and homocysteate failed to produce a specific labeling of synaptic vesicle clusters in reticulospinal or dorsal column axons. In conjunction with previous demonstrations of a stimulus-induced vesicle depletion in giant reticulospinal synapses (Wickelgren et al., 1985), these results imply that glutamate is released from synaptic vesicles. The different extravesicular glutamate levels in reticulospinal axons and dorsal column axons may relate to the different requirements for the refilling of synaptic vesicles in these functionally divergent neurons.