Recently, we have shown that by releasing specific nucleotidases the sympathetic nerves of the guinea pig vas deferens may regulate the metabolism of extracellular adenine nucleotides and consequently, the inactivation of neurotransmitter ATP. Based on the evidence for tetrodotoxin sensitivity and calcium dependence of the nerve stimulation-evoked overflow of enzyme activity, we have suggested that soluble nucleotidases may be stored in synaptic vesicles within the sympathetic nerves and released upon arrival of nerve action potentials by a mechanism similar to that for release of neurotransmitters. To further test this hypothesis we studied the time course of nerve stimulation-evoked overflow of ATP, norepinephrine (NE), releasable ATPase (r-ATPase) activity, and releasable AMPase (r-AMPase) activity under control conditions and in the presence of drugs known to selectively modulate sympathetic neurotransmission. The results show that the time course of overflow of r-ATPase and r-AMPase activities resembles the transient pattern of overflow of ATP but not the tonic pattern of overflow of NE. Vasa deferentia dissected from animals treated with reserpine release ATP, r-ATPase, and r-AMPase, whereas the overflow of NE is completely abolished. Guanethidine, on the other hand, inhibits equally well the overflow of the two neurotransmitters and the releasable nucleotidase activities. Agonists of the alpha(2)-adrenergic receptors abolish the overflow of ATP, r- ATPase, and r-AMPase but not the overflow of NE. This evidence supports the idea that the sympathetic nerves of the guinea pig vas deferens store and release ATP together with specific nucleotidases responsible for the inactivation of this neurotransmitter.