Increasing the number of transcription factor binding sites within a construct can enhance expression. In an attempt to create a synthetic locus control region for mammary expression, we have generated beta-lactoglobulin-reporter constructs with multiple copies of the cluster of transcription sites normally located within the proximal promoter. These constructs were functionally tested by stable transfection of mammary epithelial cells in vitro and in transgenic mice in vivo. Rather than enhancing expression, multimerisation of the promoter region acted neither in vivo nor in vitro to enhance expression. Indeed, its presence reduced expression. This failure to enhance expression was reflected in the inability of this region to form a DNaseI hypersensitive site autonomously in mammary chromatin in vivo. It is implicit from our study that not all combinations of transcription factor binding sites will enhance transcription.