GABAA receptors can be directly activated and potentiated by the intravenous anesthetic propofol. Previous photolabeling, modeling, and functional data have identified two binding domains through which propofol acts on the GABAA receptor. These domains are defined by the β(M286) residue at the β"+"-α"-" interface in the transmembrane region and the β(Y143) residue near the β"-" surface in the junction between the extracellular and transmembrane domains. In the ternary receptor, there are predicted to be two copies of each class of sites, for a total of four sites per receptor. We used β2α 1 γ2L and β2α1 concatemeric constructs to determine the functional effects of the β(Y143W) and β(M286W) mutations to gain insight into the number of functional binding sites for propofol and the energetic contributions stemming from propofol binding to the individual sites. A mutation of each of the four sites affected the response to propofol, indicating that each of the four sites is functional in the wild-type receptor. The mutations mainly impaired stabilization of the open state by propofol, i.e., reduced gating efficacy. The effects were similar for mutations at either site and were largely additive and independent of the presence of other Y143W or M286W mutations in the receptor. The two classes of sites appeared to differ in affinity for propofol, with the site affected by M286W having about a 2-fold higher affinity. Our analysis indicates there may be one or two additional functionally equivalent binding sites for propofol, other than those modified by substitutions at β(Y143) and β(M286).