1. The superficial abdominal flow flexor muscle was isolated from the crayfish (Cambarus clarkii) and placed in a bath solution of 100 microliters. The concentration of glutamate in this solution was measured by mass fragmentography using a gas chromatograph-mass spectrometer. 2. The excitatory post-synaptic potential (e.p.s.p.) of the slow flexor muscle and its sensitivity to L-glutamate were similar to those observed in the opener muscle of the dactyl in the walking leg or claw of the crayfish. 3. The background efflux of glutamate during control rest periods was about 20 p-mole/10 min. Nerve stimulation caused a significant increase in the efflux of glutamate. The net release of glutamate above the background was 11.9 p-mole/100 microliters. at 10 Hz stimulation and 21.1 p-mole/100 microliters. at 20 Hz stimulation. 4. When the amplitude of e.p.s.p. was decreased by streptomycin, thereby reducing the muscle contraction, the net release of glutamate by nerve stimulation was not changed. Streptomycin depressed the e.p.s.p. by its action on the post-synaptic membrane. 5. When the external concentration of Ca was lowered, the amplitude of e.p.s.p. and the net release of glutamate were decreased. 6. It is concluded that L-glutamate is released from the nerve terminals of the crayfish neuromuscular junction.