Neuroactive steroids can modulate brain excitability by interaction with several neurotransmitter receptor-associated channels. These compounds may thus exert profound influences on excitotoxic injury, i.e. neuronal cell death triggered by over-activation of glutamate receptors. It has been reported that pregnenolone sulphate (PS) and pregnenolone hemisuccinate (PHS) augment N-methyl-D-aspartate (NMDA) neurotoxicity in rat cultured neurons. Here we show that the effects of neuroactive steroids on AMPA cytotoxicity display features distinct from those on NMDA cytotoxicity. Concomitant application of PS (30-300 mu M) attenuated, rather than augmented, AMPA neurotoxicity in cortical slice cultures in a concentration-dependent manner, whereas various other steroids including pregnenolone and PHS had no effect. Inhibition of steroid sulphatase by estrone-3-O-sulphamate led to a shift of the minimum effective concentration of PS against AMPA cytotoxicity from 30 to 10 mu M. The protective action of PS was not affected by inhibition of protein synthesis or by blockade of glucocorticoid receptors, GABA(A) receptors or sigma-receptors. In dissociated cortical neurons, PS attenuated AMPA-induced inward currents whereas pregnenolone and PHS exhibited no significant effect. Thus, with strict structural specificity, PS but not pregnenolone or PHS attenuates AMPA cytotoxicity, probably by inhibiting activities of AMPA receptor-associated channels.