Abstract Two mutations of the rat serotonin 5-HT 2A receptor were made, expressed and examined for their ability to bind and be stimulated by certain tryptamines as well as their ability to bind antagonists. Mutation of Ser 207 to an Ala (S207A) resulted in no substantial changes in binding of either 5-HT 2A antagonists or agonists. In contrast, mutation of Ser 239 to an Ala (S239A) resulted in significant changes in the 5-HT 2A receptor with some but not all agonists and antagonists examined. Specifically, 5-HT had decreased affinity for the S239A mutated 5-HT 2A receptor, showing over a 10-fold decrease in receptor-binding displacement, while still being capable of stimulating IP 3 formation. However, the agonists tryptamine, 5-methoxytryptamine (5-MeOT), and N-1-isopropyl-5-methoxytryptamine; and the antagonists ketanserin, LY 86057, and LY 53857 were significantly less affected by a S239A mutation. These results suggest that while 5-HT might have a direct interaction with the Ser 239 of the 5-HT 2A receptor, tryptamine and 5-MeOT interact with this receptor in a different manner.