Several cis-regulatory DNA elements are present in the 5' upstream regulatory region of the enkephalin gene (ENK) promoter. To determine their role in conferring organ-specificity of ENK expression in mice and to circumvent the position effects from random gene insertion that are known to often frustrate such analysis in transgenic mice, we used a Cre-mediated gene knock-in strategy to target reporter constructs to a "safe haven" loxP-tagged locus in the hypoxanthine phosphoribosyltransferase (HPRT) gene. Here we report reliable and reproducible reporter gene expression under the control of the 5' upstream regulatory region of the mouse ENK gene in gene-modified mice using this Cre-mediated knock-in strategy. Comparison of two 5'ENK regulatory regions (one with and the other without known cis-regulatory DNA elements) in the resulting adult mice showed that conserved far-upstream cis-regulatory DNA elements are dispensable for correct organ-specific gene expression. Thus the proximal 1.4 kb of the murine ENK promoter region is sufficient for organ-specificity of ENK gene expression when targeted to a safe-haven genomic locus. These results suggest that conservation of the far-upstream DNA elements serves more subtle roles, such as the developmental or cell-specific expression of the ENK gene.