The aim of the present study was to investigate the effects of excitotoxic damage of the serotonergic cell bodies in the median raphe nucleus (MRN) on the scopolamine-induced working memory deficits in a single-trial light/dark inhibitory avoidance task. Rats were given 1 mg/kg of scopolamine hydrobromide (intraperitonal, IP) or saline before the inhibitory avoidance training, in which initial preference to the dark compartment (escape latency) was used to measure nonmnemonic behaviors, and response latency to enter the dark compartment immediately after the shock was used to measure working memory. It was found that scopolamine significantly reduced escape latencies in sham-lesioned rats, whereas it had no effect in the rats with MRN lesions. Although MRN lesion per se did not alter response latency, it prevented scopolamine-induced decrease in this parameter. These results suggest that the antagonistic interactive processes between serotonergic projections of the MRN and the muscarinic cholinergic system modulate nonmnemonic attentional component of working memory formation in the inhibitory avoidance.