We have mutated two residues, Ala230 and Leu231, in the C-terminal portion of the third intracellular loop of the human platelet-activating factor (PAF) receptor into Glu230 and Arg231, respectively. The Leu231 --> Arg231 substitution led to two major modifications: 1) increased constitutive activity of the PAF receptor resulting in agonist-independent production of inositol phosphates and 2) increased affinity of the receptor for binding PAF (agonist) but not WEB2086 (antagonist). The L231R mutant was able to adopt at least two conformations: (i) a higher affinity state than the corresponding state of the wild-type receptor (WT), dependent on G protein coupling, and (ii) a low affinity state, higher than the one for the uncoupled WT receptor. The Ala230 --> Glu230 substitution also resulted in two major modifications: 1) unresponsiveness in terms of phosphatidylinositol hydrolysis in response to PAF and 2) a marked decrease in affinity of the receptor for binding the agonist but not the antagonist. Competition binding studies of transient receptor expression in COS-7 cells and the inability of guanosine 5 -O-(3-thiotriphosphate) to modulate the decrease in affinity of a stable A230E mutant in Chinese hamster ovary cells suggest an inherent low affinity conformation for this mutant. Alternatively, mutation of Ala230 to Gln230 suggested that the residue 230 has a fundamental effect on receptor affinity and its charge is determinant in G protein coupling of the PAF receptor. In this report, we show that substitution of two immediately adjacent residues of the PAF receptor, Ala230 and Leu231, surprisingly leads to an inactive and a constitutively active phenotype, respectively. These results further support the concept of constitutively active G protein-coupled receptors as adopting ''active'' state conformations similar to those induced by agonist binding to WT receptors.