The 5-HT3AB receptor contains three A and two B subunits in an A-A-B-A-B order. However, serotonin function at the 5-HT3AB receptor has been shown to depend solely on the A-A interface present in the homomeric receptor. Using mutations at sites on both the primary (E122) and complementary (Y146) faces of the B subunit, we demonstrate that meta-chlorophenyl biguanide (mCPBG), a 5-HT3 selective agonist, is capable of binding to and activating the 5-HT3AB receptor at all five subunit interfaces of the heteromer. Further, mCPBG is capable of allosterically modulating the activity of serotonin from these sites. While these five binding sites are similar enough that they conform to a monophasic dose – response relationship, we uncover subtle differences in the heteromeric binding sites. We also find that the A-A interface appears to contribute disproportionately to the efficacy of 5-HT3AB receptor activation.