The effects of pregnene and androstane steroids were studied on recombinant human glycine receptors (GlyRs) by whole-cell voltage-clamp electrophysiology. The 3β-sulphates of pregnenolone (PREGS) and dehydroepiandrosterone (DHEAS) inhibited GlyR currents with K I values of 2-20 μMfor different (α 1 , α 2 , α 4 and β) GlyR subunits. PREGS resulted in a parallel shift of the response curve of glycine for α 1 GlyRs. The inhibitory potencies of DHEAS relative to PREGS were decreased in transition from embryonic α 2 towards adult α 1 β GlyRs. A decreased potency of DHEAS for α 4 versus α 2 GlyRs represents the first pharmacological difference reported between these subunits. A negative charge at C3 is required for GlyR antagonism but androsterone sulphate epimers at C3 inhibited without stereoselectivity. Some point mutations of α 1 GlyRs with characteristic functional consequences did not significantly affect the inhibitory potency of PREGS. Progesterone selectively inhibited α 2 GlyRs, while PREG and its acetic ester potentiated α 1 GlyRs. Coexpression of the α subunits with the β subunit eliminated the enhancing effects of PREG and attenuated the inhibitory potencies of the neurosteroids. Based on these data we propose that neurosteroids might modulate perinatal GlyR activity and thereby influence neuronal development.