Sensitization potentiates excitability in an interneuron, the S-cell, that is critical for this form of learning in the whole-body shortening reflex of the medicinal leech. Serotonin (5HT) also increases S-cell excitability and serotonergic modulation is known to be critical for sensitization of whole-body shortening, suggesting that 5HT mediates learning-induced enhancement of S-cell excitability. In this paper, the role of 5HT in mediating sensitization-induced potentiation of S-cell excitability was examined. Potentiation of S-cell excitability by 5HT was blocked by the 5HT receptor antagonist methysergide and by intracellular injection of the G-protein inhibitor GDP--S, indicating that a metabotropic 5HT receptor was involved. Bath application of Rp-cAMP, an inhibitor of protein kinase A (PKA), blocked 5HT-induced potentiation of excitability, while db-cAMP, a cAMP analogue that activates PKA, mimicked the potentiating effects of 5HT on the S-cell. During sensitization of the shortening reflex in semi-intact preparations, methysergide and Rp-cAMP prevented learning-induced potentiation of S-cell excitability, as well as the increase in S-cell activity that normally occurs during sensitization. Furthermore, sensitization-induced increases in the shortening reflex did not occur in preparations treated with methysergide or Rp-cAMP. These results demonstrate that sensitization-induced enhancement of S-cell excitability is mediated by 5HT and suggests that these changes may contribute to this form of learning.