The responses of dentate granule cells to medial septum (MS) and perforant path (PP) stimulation were examined in urethane anaesthetized rats. MS and PP stimulation evoked an orthodromic activation of granule cells which was correlated with the negative transient of the characteristic field potential elicited from each site. The effects of electrophoretic application of acetylcholine (ACh) and glutamate (Glu) were examined on granule cells identified in this manner. The excitatory action of ACh but not that of Glu was antagonized by atropine. Glutamate diethylester (GDEE) blocked the excitation produced by Glu but not ACh. The synaptically evoked excitation elicited by MS was blocked by atropine but unaltered by GDEE whereas the PP excitatory response was blocked by GDEE and unaltered by atropine. The results of this study indicate that two discrete excitatory systems are present in the dentate gyrus of the rat: a cholinergic system originating in the medial septum and a glutamate mediated system originating in the entorhinal cortex.