||Metalloendoprotease activity was identified in retinal homogenates using a synthetic fluorogenic metalloendoprotease substrate and specific metalloendoprotease inhibitors. The requirement of metalloendoprotease activity in neurotransmitter release was examined during the depolarization-induced release of [3H]glycine from the retina of Xenopus laevis. Neurons with high affinity uptake and calcium-dependent, K+-stimulated release of glycine have been described previously in this retina. When isolated retinas preloaded with [3H]glycine are depolarized by 22 mM K+, the usual efflux of [3H]glycine is completely abolished by the metalloendoprotease inhibitor, 1,10-phenanthroline (100 micrograms/ml). The inhibition of [3H]glycine release by 1,10-phenanthroline is dose dependent; furthermore, 1,10-phenanthroline blocks release by chelating metal and not Ca2+, since addition of equimolar calcium does not alter the inhibition. The metalloendoprotease inhibitor, carbobenzoxy (CBZ)-L-phenylalanine, also prevents release, whereas the amino acid, L-phenylalanine, has no effect. Synthetic carbobenzoxy dipeptide amides which are metalloendoprotease substrates (e.g., CBZ-Gly-Leu-amide, CBZ-Ser-Leu-amide, and CBZ-Gly-Phe-amide) also prevent [3H]glycine release in a dose-dependent and reversible manner. The synthetic dipeptide CBZ-Gly-Gly-amide, however, is not a metalloendoprotease substrate and has no effect on release. The ability of synthetic dipeptides to inhibit neurotransmitter release is amino acid specific, dose dependent, reversible, and matches their ability to interact with characterized metalloendoproteases. Depolarization-stimulated transmitter release may therefore require the activity of a metalloendoprotease.