We describe, in the present work, a continuous procedure for measuring the release of glutamate, and have applied it to analyze the co-release of glutamate and acetylcholine from cholinergic nerve terminals of electric organ synaptosomes. The two substances were measured in similar conditions using the two continuous chemiluminescent assays. The protein "Mediatophore" reconstituted in proteoliposomes was also able to translocate both transmitters with a preference for glutamate when the external calcium was increased between 3 and 10 mM. However, at lower calcium concentrations a clear preference for acetylcholine was found. For a regulated calcium entry as obtained by electrical stimulation of the nerve the release mechanism was fully specific for acetylcholine. Since the specificity of mediatophore depends on the local calcium amounts, the possible role of mediatophore at acetylcholine or glutamate specific synapses may be envisaged and is discussed in relation to the calcium control of specificity.