We have cloned the mouse 5-HT6 serotonin receptor and examined structure-function relationships in the C-terminal end of the third cytoplasmic (CIII) loop, introducing point mutations by site-directed mutagenesis at positions 264 to 268. We examined the ability of 5-HT6 wild type and receptor mutants to activate a cAMP responsive reporter gene when transiently expressed in JEG-3 or COS-7 cells. The wild type 5-HT6 receptor showed strong constitutive activity even when expressed at very low levels and which increased in proportion to the amount of receptor cDNA transfected. Three of the five mutants investigated (K264I, K267A and A268R) showed reduction in constitutive activity compared to wild type. These data suggest that constitutive activity may be important to 5-HT6 receptor activity in vivo and that, unlike some other G-protein coupled receptors, alteration in the BBXXB CIII-loop motif reduces rather than further activates basal activity of the murine 5-HT6 receptor.