Potentiating neuroactive steroids are potent and efficacious modulators of the GABAA receptor that act by allosterically enhancing channel activation elicited by GABA. Steroids interact with the membrane-spanning domains of the α subunits of the receptor, whereas GABA binds to pockets in the interfaces between β and α subunits. Steroid interaction with a single site is known to be sufficient to produce potentiation, but it is not clear whether effects within the same β-α pair mediate potentiation. Here, we have investigated whether the sites for GABA and steroids are functionally linked (i.e., whether the occupancy of a steroid site selectively affects activation elicited by GABA binding to the transmitter binding site within the same β-α pair). For that, we used receptors formed of mutated concatenated subunits to selectively eliminate one of the two GABA sites and one of the two steroid sites. The data demonstrate that receptors containing a single functional GABA site are potentiated by the neurosteroid allopregnanolone regardless of whether the steroid interacts with the α subunit from the same or the other β-α pair. We conclude that steroids potentiate the opening of the GABAA receptor induced by either agonist binding site.