N-methyl-D-aspartate (NMDA) receptors (NMDARs) are ligand-gated ion channels belonging to the family of ionotropic glutamate receptors. Cell lines stably expressing recombinant NMDARs of predetermined composition provide a feasible system in identifying selective agonists and antagonists of this receptor and elucidating its related signaling pathway. A human embryonic kidney (HEK) 293 based cell line stably expressing rat NR1b NMDAR subunit was developed employing a doxycycline-inducible gene expression system. HEK293 cells expressing the regulatory vector pTet-On-Advanced (HEK293 Tet-On Advanced cells, Clontech) were transfected with rat NR1b cDNA cloned in the pTRE-Tight doxycycline-inducible vector, along with the hygromycin resistant marker. Inducible expression of NR1b subunit in the hygromycin resistant clone designated HEK293 Tet-On Advanced/rNR1b was tested structurally and functionally by reverse transcription (RT)-PCR, immunofluorescence staining, electrophysiological recording, and Ca2+ imaging. Inhibition by conantokin-T and conantokin-G on NR1bR2A and NR1bR2B combination of NMDARs was also examined. The establishment of this cell line provided the possibility of stably expressing different combinations of NMDAR subunits using Tet-On expression system for NMDAR related study in the future.