The effect of the neurosteroid pregnenolone sulfate (PS) on the glycine receptor-mediated response was studied in cultured chick spinal cord neurons using the whole-cell voltage-clamp recording technique. PS rapidly and reversibly inhibits the glycine-induced current in a dose-dependent manner, with an EC50 of 3.7 microM and a maximal inhibition of 100%. The fact that antagonism of the glycine response by PS is neither voltage- nor agonist-dependent indicates that PS does not act as an open-channel blocker. Furthermore, inhibition by PS of the glycine-induced current appears to be of a competitive type since the drug induces a parallel, rightward shift of the glycine dose-response curve.