We have cloned the 5′-region of the murine N-methyl-d-aspartate (NMDA) receptor channel subunit NR2C (GluRε3) gene and characterized the cis- and trans-activating regulatory elements responsible for its tissue specific activity. By using a native ε3-promoter/lacZ-construct & various 5′-deletion constructs, we compared β-galactosidase expression in non-neuronal NIH3T3 cells and in neuronal ε3-gene-expressing HT-4 cells and show that large parts of the ε3 promoter are responsible for the repression of the ε3 gene in non-neuronal cells. Deletion of exon 1 sequences led to an enhancement of ε3 transcription, suggesting a role of the 5′-untranslated region in ε3 gene regulation. Sequence analysis of the promoter region revealed potential binding sites for the transcription factor Sp1, the murine fushi tarazu factor1 (FTZ-F1) homologues, embryonic LTR binding proteins (ELP1,2,3) and steroidogenic factor (SF-1), as well as for the chicken ovalbumin upstream promoter transcription-factor (COUP-TF). Electrophoretic mobility shift assays confirmed specific binding of Sp1, SF-1 and COUP-TFI. Whereas point mutation studies indicate that, in neuronal HT-4 cells, Sp1 is apparently not critically involved in basal ε3 gene transcription, SF1 is a positive regulator. This was evident from a selective enhancement of ε3-promoter-driven reporter gene expression upon cotransfection of an SF1-expression vector, which was reverted by deletion and point mutation of the SF1 binding site.