The γ-aminobutyric acid type A (GABAA) receptor is modulated by a number of neuroactive steroids. Sulfated steroids and 3β-hydroxy steroids inhibit, while 3α-hydroxy steroids typically potentiate the receptor. Here, we have investigated inhibition of the α1β3γ2L GABAA receptor by the endogenous neurosteroid 3α-hydroxy-5β-pregnan-20-one (3α5βP) and the synthetic neuroactive steroid 3α-hydroxy-5α-androstane-17β-carbonitrile (ACN). The receptors were expressed in Xenopus oocytes. All experiments were done using two-electrode voltage-clamp electrophysiology. In the presence of low concentrations of GABA, 3α5βP and ACN potentiate the GABAA receptor. To reveal inhibition, we conducted the experiments on receptors activated by the combination of a saturating concentration of GABA and propofol to fully activate the receptors and mask potentiation, or on mutant receptors in which potentiation is ablated. Under these conditions, both steroids inhibited the receptor with IC50s of ∼13 μM and maximal inhibitory effects of 70-90%. Receptor inhibition by 3α5βP was sensitive to substitution of the α1 transmembrane domain (TM) 2-2' residue, previously shown to ablate inhibition by pregnenolone sulfate. However, results of coapplication studies and the apparent lack of state dependence suggest that pregnenolone sulfate and 3α5βP inhibit the GABAA receptor independently and through distinct mechanisms. Mutations to the neurosteroid binding sites in the α1 and β3 subunits statistically significantly, albeit weakly and incompletely, reduced inhibition by 3α5βP and ACN. SIGNIFICANCE STATEMENT: The heteromeric GABAA receptor is inhibited by sulfated steroids and 3β-hydroxy steroids, while 3α-hydroxy steroids are considered to potentiate the receptor. We show here that 3α-hydroxy steroids have inhibitory effects on the α1β3γ2L receptor, which are observed in specific experimental settings and are expected to manifest under different physiological conditions.