The mouse κ-opioid receptor (KOR) gene is expressed in mouse embryonal carcinoma P19 cells and induced by retinoic acid (RA) within 24 h. An RA-responsive cis-acting element is identified within promoter I of the KOR gene. This element contains a GC box, a putative binding site for transcription factor Sp1. Enhanced binding of Sp1 to this GC box correlates with RA induction of KOR gene. Phosphatase inhibitor (sodium pyrophosphate) decreases RA induction of this promoter, whereas hypophosphorylation of Sp1 results in an increase in its DNA binding affinity to this promoter as demonstrated by in vitro gel retardation and in vivo chromatin immunoprecipitation assays. Consistently, the inhibitor of MEK, PD98058, dose-dependently enhances RA induction of this promoter, suggesting that the ERK signaling pathway is negatively involved in the RA induction of mouse KOR gene activities. Collectively, enhanced binding of Sp1 to promoter I of the KOR gene as a result of inhibiting the ERK pathway contributes to RA induction of this gene in P19.