The tissue specificity conferred by cis‐acting regulatory elements of the rat insulin 1 gene was examined in both cultured cells and transgenic mice. The enhancer region (–346/–103) coupled to a ubiquitous promoter activated expression of a reporter gene in insulinoma cells but not in fibroblasts, in agreement with our previous work, and the specific expression was limited to a subregion containing the FAR and FLAT elements (–252/–199). In transgenic mice, however, this FAR‐FLAT minienhancer alone failed to activate a reporter gene. Under the same conditions, in vivo, the enhancer (–346/–103) activated gene expression, but did not confer complete pancreatic specificity. The transgene, in this case, was expressed in pancreas and also in brain. Reassociation of the rat insulin 1 promoter (–102/+9) with the enhancer (–346/–103) prevented expression in brain and thus restored pancreatic specificity. All of these observations indicate that tissue‐specific expression of the rat insulin 1 gene, in vivo, results from interaction of multiple sequence elements and not from any single minimal sequence.