1. This study describes the in vitro characterization of two potent and selective 5-HT6 receptor antagonists at the rat and human recombinant 5-HT6 receptor. 2. In binding assays with [3H]-LSD, 4-amino-N-(2,6 bis-methylamino-pyrimidin-4-yl)-benzene sulphonamide (Ro 04-6790) and 4-amino-N-(2,6 bis-methylamino-pyridin-4-yl)-benzene sulphonamide (Ro 63-0563) had mean pKi values +/-s.e.mean at the rat 5-HT6 receptor of 7.35+/-0.04 and 7.83+/-0.01, respectively and pKi values at the human 5-HT6 receptor of 7.26+/-0.06 and 7.91+/-0.02, respectively. 3 .Both compounds were found to be over 100 fold selective for the 5-HT6 receptor compared to 23 (Ro 04-6790) and 69 (Ro 63-0563) other receptor binding sites. 4. In functional studies, neither compound had any significant effect on basal levels of cyclicAMP accumulation in Hela cells stably expressing the human 5-HT6 receptor, suggesting that the compounds are neither agonists nor inverse agonists at the 5-HT6 receptor. However, both Ro 04-6790 and Ro 63-0563 behaved as competitive antagonists with mean +/-s.e.mean pA2 values of 6.75+/-0.07 and 7.10+/-0.09, respectively. 5. In rats habituated to observation cages, Ro 04-6790 produced a behavioural syndrome similar to that seen following treatment with antisense oligonucleotides designed to reduce the expression of 5-HT6 receptors. This behavioural syndrome consisted of stretching, yawning and chewing. 6. Ro 04-6790 and Ro 63-0563 represent valuable pharmacological tools for the identification of 5-HT6 receptors in natural tissues and the study of their physiological function.