There is considerable evidence that the N-methyl-D-aspartate receptor (NMDAR) is a component of excitatory amino acid synapses in the ascending auditory pathway. The availability of mice that are defective in NMDAR epsilon 1 or NMDAR epsilon 4 subunit paves the way for investigations on the role of this receptor in auditory function. Non-radioactive in situ hybridization was used in the parent C57/6J wild strain to determine if these subunits are normally expressed in cochlear nucleus (CN) and superior olivary complex (SOC) and to confirm their absence in the respective mutant mice. Evoked auditory brainstem response (ABR) to normal acoustic stimulation was investigated to assess function. In situ hybridization revealed the expression of NMDAR epsilon 1 and epsilon 4 subunits mRNAs in major neuronal types in the CN and SOC of the wild type mice while epsilon 1 and epsilon 4 expression were absent in their respective mutant mice. The ABR threshold for the epsilon 1 mutant mice was similar to that of wild type mice however the threshold for the epsilon 4 mutant mice was significantly elevated. These results suggest a role for the NMDAR epsilon 4 in normal auditory functions while the NMDAR epsilon 1 may have a less critical function under normal conditions.