Recent methods allow the study of neurons that contain zinc in synaptic vesicles of their boutons (Timm-stainable boutons) by the intravital precipitation (local or throughout the CNS) of the vesicular zinc with selenium compounds and its subsequent retrograde transport to the parent neurons, where the precipitate can be silver enhanced. The present study is a description of the distribution of zinc-containing neurons, their possible connections and their terminal fields within the hippocampal region of the rat. Problems inherent to the methods are addressed. Finally, based on the results and a review of literature, the possible function of zinc in the hippocampal region is considered. Neurons which contain silver-enhanced precipitates were observed in layers II, V and VI of the lateral entorhinal area and in layers V and VI of the medial entorhinal area. In the parasubiculum, labeled cells were seen in layer II/III of the parasubiculum a and in layer V. Labeled cells in the presubiculum were concentrated in layers III and V, in the hippocampal pyramidal cell layer and the dentate granule cell layer, but neurons containing precipitates were largely absent from the subiculum. Zinc-containing axonal boutons defined subpopulations within principal hippocampal neuron populations. Within layer II of the lateral entorhinal cortex and the pyramidal cell layer for regio inferior deeply situated neurons were labeled, whereas superficially placed pyramidal cells were labeled in regio superior. The neuropil staining described in the present study corresponded to that found in earlier studies. However, glial and vascular staining or unspecific background were largely absent, and the neuropil staining could unequivocally be identified light microscopically. Methodological problems are most prominently reflected in unstained mossy fibers in some animals. Based on series from animals treated with decreasing doses of sodium selenite and increased survival times, this problem can be related to small amounts of circulating reactive selenium and a competition of zinc compartments (vesicles) for the selenium. Staining will fail where the competition prevents individual compartments from reaching a threshold amount of zinc precipitate for silver amplification. A guide to evaluate histological material is provided. The distribution of zinc-containing boutons and their cells of origin indicate that zinc-containing and zinc-negative projections are not organized as parallel pathways. The mossy fibers provide an example of a pure zinc-containing pathway. Projections from regio superior to the dorsal presubiculum are likely to be zinc-negative while projections from the same area to the subiculum are zinc-containing.(ABSTRACT TRUNCATED AT 400 WORDS)