1. The levels of adenine nucleotides and adenosine which accumulate in the neuromuscular junction during nerve stimulation of the rat extensor digitorum longus (EDL) muscle were assayed biochemically. The sources were also determined by the use of different inhibitors. 2. ATP and total adenine nucleotide release increased as stimulation frequency increased, consistent with previous evidence indicating ATP release from presynaptic sources. 3. Adenosine levels also increased during nerve stimulation. However, accumulation decreased by 46-58% when muscle activation was blocked by the addition of d-tubocurarine (dTC). Adenosine levels also decreased by 40-59% when adenine nucleotide hydrolysis to adenosine was blocked by the addition of 1 mM-alpha,beta-methyladenosine 5'-diphosphate. Thus, approximately half of the extracellular adenosine is released from activated muscle while the other half is derived from adenine nucleotide hydrolysis. 4. Similar quantities of adenine nucleotide and acetylcholine (ACh) accumulated during nerve stimulation. With adenine nucleotide and ACh hydrolysis blocked by alpha,beta-methyladenosine 5'-diphosphate and eserine, respectively, the calculated amounts of adenine nucleotide and ACh released were 1.2 x 10(-16) and 1.5 x 10(-16) mol (stimulus impulse)-1 endplate-1. 5. AH5183 (vesamicol), which blocks ACh release, reduced extracellular ACh and adenine nucleotide accumulation by 40 and 45%, respectively. It did not affect adenosine release from the activated muscle. 6. Theophylline (100 microM), which blocks adenosine receptors, caused ATP accumulation to increase by 38%; extracellular levels of adenosine derived from adenine nucleotide hydrolysis also increased by 17%. These results are consistent with the presence of adenosine-mediated inhibition of adenine nucleotide release. 7. It is concluded that adenine nucleotides (presumably in the form of ATP) and ACh are released jointly, and that ATP is hydrolysed fairly rapidly to adenosine. Adenosine resulting from ATP hydrolysis accounts for about half of the extracellular adenosine accumulating during nerve stimulation, while the other half is released directly by the underlying muscle.