Using rapid agonist applications to transfected HEK-293 cells, we investigated pregnenolone sulfate (PS) effects on deactivation and desensitization of recombinant NMDA receptors subtypes. PS prolonged the deactivation of responses produced by brief applications of L-glutamate with all subunit combinations tested. The action of PS was larger on NR1a/NR2A than on NR1a/NR2B channels. PS slowed the rate of macroscopic desensitization of the responses with all subunit combinations tested. In contrast, PS had little effect on current rise time and had much reduced action on responses with L-cysteate, a low affinity agonist. Our results suggest that PS decreases agonist unbinding. However, this action is counteracted by decreased desensitization. Since desensitization produces slow deactivating components, particularly with NR1a/NR2B receptors, this underlies the decreased PS effect with these subtypes. Indeed PS action was mainly observed on the fast component of deactivation. Furthermore, prolongation of NR1a/NR2A responses was similar to that of responses from NR1b/NR2B receptor, a subtype characterized by reduced desensitization. PS prolongation of evoked NMDA receptor mediated synaptic currents from cortical neuronal primary culture(s) was not significantly different from that of responses with NR1a/NR2B receptors indicating that native receptors in these neurons comprised at least some heteromeric combinations of these two subunits.