Levels of ATP and related adenylates were measured in the terminal region of efferent nerves in the crayfish opener muscle using the luciferin-luciferase method. Following 1 min of stimulation at 50 Hz, the average (+/- SE) ATP content rose from 13.4 (+/- 1.5) to 19.0 (+/- 2.1) nmol/mg dry weight. The amounts of ADP, AMP, and the phosphagen phosphoarginine did not change significantly. Thus, the increased ATP was not derived from any of these potential sources. The increase was found to depend on synaptic activation, however, for its magnitude was directly related to the concentration of extracellular Ca2+, and it was blocked when CoCl2, verapamil, ruthenium red, or gamma-methylglutamate and picrotoxin were added to the bath. Addition of ATP to the bath solution also increased nerve ATP levels. Based upon measurements of sucrose distribution, only 50% of this increase was in the extracellular water space. The remainder of the ATP had either entered the nerve, become adsorbed extracellularly, or both. Addition of 2-deoxy-D-glucose and gamma-32P-ATP to the bath resulted in the formation of 32P-2-deoxy-D-glucose-6-P by the nerve. This suggests that a fraction of the extracellular ATP does enter the neuron chemically intact. To determine whether exogenous ATP is the source of the increased ATP measured in the nerve following stimulation, the bath was assayed for ATP. Stimulation did cause ATP levels to increase significantly; however, the maximum concentration was 3 orders of magnitude lower than that required to increase ATP levels in resting nerve.(ABSTRACT TRUNCATED AT 250 WORDS)