PSD-95 proteins are the most abundant scaffold proteins in the postsynaptic density. PDZ1, PDZ2, and PDZ3 domains (PDZ1-3) are the major components of PSD95. PDZ domains bind to the carboxyl-terminus of PSD-95 partnering receptors, such as NMDA and AMAP receptors. Anesthetics were found to perturb the interaction between PDZ domains and their target proteins. However, it was unclear whether anesthetics interact directly with the PDZ domains. Using NMR, we have investigated anesthetics’ effects (such as interaction region and affinity) on the PDZ1-3 domains of PSD-95. 80% of the residues of PDZ123 could be identified with high resolution NMR. Anesthetics (halothane, isoflurane, and sevoflurane) were titrated into the PDZ1-3 samples at 4 ∼ 5 different concentrations, which were determined using 19F NMR with trifluoroacetic acid as the external standard. Certain residues showed chemical shift changes in an anesthetic concentration dependent manner in the 15N-1H HSQC spectra. The combined chemical shifts change of 15N and 1H quantified anesthetic perturbation. We found that all three tested anesthetics affected PDZ1-3, but PDZ2 had the most affected residues, while PDZ3 had the least. The affected residues in PDZ1 and PDZ2 were mostly in, or close to, their peptide binding groove, whereas very few residues close to the binding groove in PDZ3 were affected. These findings are valuable for understanding how anesthetics alter signal transduction by perturbing PSD, in particular PDZ1-3 domains. The results are useful for the study of anesthetic perturbation on peptide binding on the PDZ domain with atomic resolution. Supported by NIH (NOT-GM-08-130 and R01GM056257-11 ).