The construction of mitotically stable yeast strains for heterologous gene or pathway expression often requires chromosomal integration. However, transcription levels vary between different chromosome regions. We therefore characterized 20 different integration sites of the Sacchromyces cerevisiae genome by inserting lacZ as a reporter gene under the control of two different promoters and determining expression levels through enzyme activity measurement. An up to 8.7-fold difference was detected between the sites conferring lowest and highest expression, respectively. This opens the opportunity for modulating gene expression levels while retaining promoter and culture conditions. Copyright © 2009 John Wiley & Sons, Ltd.