Experiments were conducted to examine the effects of reducing the activity of 5-hydroxytryptamine (5-HT) containing neurons originating in the dorsal raphe (DR) or median raphe (MR) on behavioural inhibition resulting from non-reward. Groups of rats equipped with guide cannulae aimed at either the DR or MR were trained to bar press for food reward in three different operant paradigms. The 5-HT agonist 8-OH-DPAT was then infused into either the DR or MR to suppress the activity of 5-HT neurons and the effects on response inhibition resulting from the omission of reward were measured. At doses of 0.2 and 1 microgram 8-OH-DPAT injected into the MR increased responding during extinction of a continuously reinforced response. Similar injections into the DR failed to alter responding. In an omission training paradigm, requiring animals to withhold responding for a period of at least 20 s to receive free reward, rats treated with 1 microgram 8-OH-DPAT in the MR showed significantly higher levels of responding. Normal inhibition was observed following DR injections of 8-OH-DPAT. In a third paradigm both DR and MR injections of 8-OH-DPAT impaired accuracy of responding in a signalled go/nogo successive discrimination. In the case of DR treatment this resulted from a decrease in responding during reward periods signalled by S+. However, 5 micrograms 8-OH-DPAT in the MR significantly increased responding during periods of non-reward, signalled by S-. The pattern of results across these paradigms shows that reducing 5-HT activity at the level of the MR results in a failure to demonstrate normal behavioural inhibition induced by non-reward. These results support the notion that MR 5-HT neurons may be involved in controlling behavioural inhibition by detecting signals of non-reward, which then act to suppress ongoing behaviour. Serotonergic neurons arising from the DR do not appear to be involved in mediating behavioural inhibition resulting from omission of reward.