Abstract: Progesterone and its A-ring reduced metabolites are allosteric activators of GABAA receptors. The studies reported here examined the effects of these steroids on brain nicotinic receptors using an 86Rb+ efflux assay that likely measures the function of α4β2-type nicotinic receptors and [3H]dopamine release, which may be modulated by an α3-containing nicotinic receptor. Both of the A-ring reduced metabolites of progesterone were noncompetitive inhibitors of both assays, whereas progesterone inhibited only the 86Rb+ efflux assay. The 86Rb+ efflux assay was slightly more sensitive than was the dopamine release assay to steroid inhibition. Inhibition developed slowly for both assays (t1/2 = 0.4 min) and was reversed even more slowly (t1/2 = 10–15 min). Steroid addition did not alter either the rate of association of [3H]nicotine binding to brain membranes, nor was equilibrium binding changed. These findings argue that neurosteroids are allosteric inhibitors of brain nicotinic receptors.