Separate groups of rats were trained to discriminate either 0.1 mg/kg (low dose; L) or 2.5 mg/kg (high dose; H) of 8-OH-DPAT from saline, in a standard operant task. Both cues were found to be dose, time and route dependent and generalized completely to the 5-HTu agonists ipsapirone and flesinoxan. Buspirone substituted completely for 8-OH-DPAT in L and partially in H, whereas the 5-HT]A,1B receptor agonist eltoprazine substituted completely for 8-OH-DPAT in H but only partially in L. The 5-HT1A/1B receptor agonist RU24969, the 5-HTIB/2chA receptor agonist TFMPP and the 5-HT reuptake blocker fluvoxamine did not completely mimic the effect of 8-OH-DPAT in either L or H and the 5-HT,A mixed agonists/antagonists BMY 7378 and NAN-190 produced partial generalization in L, but no generalization in H. In antagonism tests, NAN-190 and BMY 7378 only partially blocked the 8-OH-DPAT cue in both groups. The non-selective 5-HT receptor antagonist methysergide did not completely block the 8-OH-DPAT cue in L or H. However, in generalization studies, it completely mimicked the 8-OH-DPAT cue in L and produced partial generalization in H. The /8-adrenergic/5-HTIA/1B receptor antagonist pindolol completely blocked the 8-OH-DPAT cue in L and H and did not mimic the 8-OH-DPAT cue in either condition. The a2-adrenoceptor blocker yohimbine substituted fully for the 8-OH-DPAT cue in L and partially in H. Idazoxan did not substitute for the cue of 8-OH-DPAT in H, but produced nearly 80% generalization in L. The dopamine receptor antagonist pimozide neither blocked nor mimicked the cue of 8-OH-DPAT in either group. A number of other drugs (i.e. m-CPP, S (-propranolol, DOI, ketanserin, clonidine and apomorphine) were only tested in H. S( - (-Propranolol blocked the 8-OH-DPAT cue but the other compounds produced neither stimulus generalization nor antagonism. The present study demonstrates that the cues produced by the low and the high training dose of 8-OH-DPAT are quantitatively different and mediated by the agonistic activity of 8-OH-DPAT at 5-HT1A receptors. Although the results suggest that the 8-OH-DPAT cue (both L and H) is mediated via postsynaptic 5-HT,A receptors, the involvement of presynaptic 5-HT,A receptors cannot yet be ruled out.