The release of endogenous zinc was studied in the hippocampus of the anesthetized rat. Push-pull cannulae were bilaterally introduced in the hippocampus and zinc concentrations determined by atomic absorption spectrophotometry. We first studied the regional distribution of K+-evoked release of zinc. A 2 min (30 mM) K+ pulse produced a release of endogenous zinc, when the push-pull cannulae were located in the vicinity of the mossy fibers (CA3 or hilus) but not in other regions (including CA1, fimbria, molecular layer of the fascia dentata, thalamus etc...). In CA3 the maximal release was of 2000 ng/ml (200 times the levels of zinc present in the cerebrospinal fluid (CSF)). Destruction of the mossy fibers by a local unilateral injection of 1.5 micrograms of colchicine dissolved in 0.6 microliter of a saline solution, eliminated this release without affecting the release in the control (contralateral) side. Electrical stimulation of the perforant path at 1 Hz did not evoke a release of zinc. In contrast at 10 Hz this stimulation produced a burst of population spike and a significant release of zinc in the mossy fibers (and not in other regions of the hippocampus). These experiments provide direct evidence that zinc is selectively released from the mossy fibers.