N-methyl-D-aspartate receptors play a critical role in synaptogenesis, synaptic plasticity, and excitotoxicity. They are heteromeric complexes of NR1 combined with NR2A-D and/or NR3A-B subunits. The subunit composition determines the biophysical and pharmacological properties of the N-methyl-D-aspartate receptor channel complex. In this study, we report that responses mediated by recombinant rat N-methyl-D-aspartate receptors expressed in human embryonic kidney HEK293 cells are differentially affected by naturally occurring neurosteroid pregnenolone sulfate. We show that responses induced by 1mM glutamate in NR1-1a/NR2A and NR1-1a/NR2B receptors are potentiated five- to eight-fold more by pregnenolone sulfate than responses of NR1-1a/NR2C and NR1-1a/NR2D receptors with no differences in the concentration of pregnenolone sulfate that produced 50% potentiation. In addition to potentiation, pregnenolone sulfate also has an inhibitory effect at recombinant N-methyl-D-aspartate receptors, with values of the concentration of pregnenolone sulfate that produces 50% inhibition of NR1/NR2D=NR1/NR2C<NR1/NR2B<NR1/NR2A. In addition, we show that the structure of the extracellular loop between the third and fourth transmembrane domains of the NR2 subunit is critical for both the potentiating and inhibitory effects of pregnenolone sulfate. The modulatory effects of pregnenolone sulfate are consistent with a model in which this neurosteroid acts at two distinct binding sites on the N-methyl-D-aspartate receptor. These data provide insight into the mechanisms by which pregnenolone sulfate and related sulfated neurosteroids modulate activity of N-methyl-D-aspartate receptor channels.