The hippocampus is a central area of the memory-related neural system. Combined immunohistochemistry against choline acetyl transferase and retrograde transneuronal labelling of the pseudorabies virus were used to identify cholinergic neurons in the central nervous system projecting to the hippocampal formation of the rat. Five to ten µL of Bartha strain of pseudorabies virus were injected into the dentate gyrus, CA1 and CA3 of the hippocampus of 20 Sprague Dawley rats using stereotaxic instrument. Forty eight to 96 hr after the injection, the brains were removed and the tissue sections were processed for double immunofluorescence procedure using polyclonal antibodies against pseudorabies virus or choline acetyl transferase. The double labelled neurons were distributed at several different nuclei and the labelling patterns of three different areas of the hippocampus were similar. These data suggests that the cholinergic innervation to the hippocampus were distributed in a transsynaptic manner throughout the whole brain area.