The experiments investigated the interactions between median raphe nucleus (MRN) serotonergic and septo-hippocampal muscarinic cholinergic systems in the modulation of forming and storing performances of working memory. Rats with ibotenic acid-induced MRN-lesion bilaterally received scopolamine (2-4 microg/each side) infusion into the dentate gyrus of the dorsal hippocampus and were tested in a single trial step-through inhibitory avoidance. Initial preference to the dark compartment (escape latency) was taken as the measure of non-mnemonic behaviours and response latency to enter the dark compartment immediately after the foot-shock was used to measure working memory. The high-dose scopolamine infusion 10 min before the training decreased escape latencies in the sham-lesioned rats, whereas had no effect in the MRN-lesioned rats. Although MRN lesion per se did not alter response latency, it alleviated pre-training scopolamine-induced decrease, but aggravated post-training scopolamine-induced reduction in this parameter. These results suggest that the antagonistic interactive processes between MRN-serotonergic and hippocampal cholinergic systems modulate non-mnemonic component of working memory formation, whereas the storing performance of working memory is modulated by the synergistic interactions between these systems in the hippocampus, mainly in the dentate gyrus.