Previously, we reported the presence of dual (distal and proximal) promoters in mouse μ-opioid receptor (mor) gene, with mor transcription in mouse brain predominantly initiated by the proximal promoter. Sp factors, bound to double-stranded (ds) cis-regulatory elements, are critical for proximal promoter activity. Here, we further report that a single-stranded (ss) cis-regulatory element and trans-acting protein factor are also important for proximal promoter activity. A 26-bp mor polypyrimidine/polypurine region (PPy/u) can adopt ss DNA conformation, as demonstrated by S1 nuclease sensitivity. Using electrophoretic mobility shift analysis with nuclear extracts from mor-expressing SH-SY5Y cells, we demonstrate that the sense strand of PPy/u interacts with a major nuclear protein, termed mor polypyrimidine-binding protein (mPy), which is not related to Sp factors. Southwestern blot analysis indicated that mPy protein is ∼25 kDa in size. Functional analysis suggests that mPy protein can trans-activate mor promoter as well as a heterologous promoter. Moreover, combinatorial activation of ss (mPy) and ds (Sps) DNA binding factors, interacting with an overlapping DNA (PPy/u) region, is necessary for proximal promoter activation. Thus our results suggest that transcription of mouse mor gene is regulated by an interplay of ss and ds DNA binding factors.