While the highest expression level of 5-HT(7) receptors in the brain is observed in intralaminar and midline thalamic neurones, the physiological role of these receptors in this structure is unknown. In vivo recordings have shown that stimulation of the serotonergic raphe nuclei can alter the response of these neurones to a nociceptive stimulus, suggesting that serotonin modulates their firing properties. Using the patch-clamp technique in rat thalamic brain slices, we demonstrate that activation of 5-HT(7) receptors can strongly modulate the excitability of intralaminar and midline thalamic neurones by inhibiting the calcium-activated potassium conductance that is responsible for the slow afterhyperpolarization (sAHP) following a spike discharge. This sAHP was inhibited after activation of the cAMP pathway, either by bath application of forskolin or intracellular perfusion with 8-bromo-cAMP. The inhibitory effect of 5-HT(7) receptors on sAHPs was blocked by the protein kinase A antagonist R(P)-cAMPS. Calcium-imaging experiments showed no change in intracellular calcium levels during the 5-HT(7) response, indicating that in these neurones, a global calcium signal was not necessary to activate the cAMP cascade. Finally, bath application of serotonin produced a strong increase in cytosolic cAMP concentration, as measured using the fluorescent probe FlCRhR, and an inhibition of the sAHP. Taken together, these results suggest that 5-HT(7) receptors are implicated in the effect of 5-HT on sAHP in intralaminar and midline thalamic neurones, an effect that is mediated by the cAMP second-messenger cascade.