Synaptic glutamate release involves the accumulation of cytoplasmic glutamate in synaptic vesicles, whereafter it is released by triggered exocytosis. As glutamatergic terminals are known to be functionally diverse it was of interest to examine whether the presynaptic glutamate supply differs between individual axon terminals with distinct release properties. The glutamatergic terminals in the crustacean neuromuscular system system comprise a “phasic” type which shows fatigue of release during repetitive stimulation, and a “tonic” type which can maintain transmission for long periods. Quantitative immunogold analysis showed that the axons in a tonic nerve innervating slow muscles in the abdomen contained two times higher levels of glutamate labeling over axoplasmic matrix and over mitochondria, as compared to the corresponding elements in a phasic nerve. Similar results were obtained when adjacent phasic and tonic axons in a mixed nerve innervating leg muscles were compared. In the terminal regions of tonic and phasic axons the glutamate labeling differed correspondingly over axoplasmic matrix and mitochondria, while the synaptic vesicles showed a similar strong accumulation of labeling in both types of terminal. The level of labeling for glutamine, a glutamate precursor, was closely similar in phasic and tonic axons. The axoplasmic glutamate concentration was estimated to be in the low millimolar range, through comparison with coprocessed conjugates with known glutamate concentration. These results show that fatigue-resistant tonic axons and terminals contain higher levels of glutamate than fatiguable phasic axons, presumably representing an adaptation to the markedly different impulse activities in the two types of neuron. The axonal glutamate concentrations are in the range of the Km value for vesicular glutamate transport. Thus in tonic axons the high glutamate level appears to promote an efficient refilling of synaptic vesicles during sustained release, while in phasic axons the refilling should be slower which is compatible with an infrequent release.