AbstractThe PSD‐95/Dlg/ZO‐1 (PDZ) domain‐containing proteins MALS and PSD‐95 localize to post‐synaptic densities and bind the COOH‐termini of NR2 subunits of the NMDA receptor. The effects of MALS‐2 and PSD‐95 on the channel activity of NMDA receptors were compared using the Xenopus oocyte expression system. Both MALS‐2 and PSD‐95 increased the current response of the NR1‐NR2B receptor to l‐glutamate. In contrast, the current response of the NR1‐NR2A receptor was increased by PSD‐95 but not by MALS‐2. MALS‐2 had no effect either on the potentiation of NR1‐NR2A or NR1‐NR2B channel activity by protein kinase C, or on Src‐mediated potentiation of NR1‐NR2A activity, whereas PSD‐95 almost completely inhibited the effects of these protein kinases. Construction of chimeras of MALS‐2 and PSD‐95 revealed that the first two PDZ domains and two NH2‐terminal cysteine residues are essential for the inhibitory effects of PSD‐95 on protein kinase C‐mediated potentiation of NR1‐NR2A and NR1‐NR2B channel activity, respectively. The second of the three PDZ domains of PSD‐95 was required for its inhibition of Src‐mediated potentiation of NR1‐NR2A activity. These results indicate that the NR1‐NR2A and NR1‐NR2B receptors are modulated differentially by MALS‐2 and PSD‐95, and that similar regulatory effects of PSD‐95 on these receptors are achieved by distinct mechanisms.