The widely used pESC vector series (Stratagene, La Jolla, CA, USA) with the bidirectional GAL1/GAL10 promoter provides the possibility of simultaneously expressing two different genes from a single vector in Saccharomyces cerevisiae. This system can be induced by galactose and is repressed by glucose. Since S. cerevisiae prefers glucose as a carbon source, and since its growth rate is higher in glucose than in galactose‐containing media, we compared and evaluated seven different promoters expressed during growth on glucose (pTEF1, pADH1, pTPI1, pHXT7, pTDH3, pPGK1 and pPYK1) with two strong galactose‐induced promoters (pGAL1 and pGAL10), using lacZ as a reporter gene and measuring LacZ activity in batch and continuous cultivation. TEF1 and PGK1 promoters showed the most constant activity pattern at different glucose concentrations. Based on these results, we designed and constructed two new expression vectors which contain the two constitutive promoters, TEF1 and PGK1, in opposite orientation to each other. These new vectors retain all the features from the pESC–URA plasmid except that gene expression is mediated by constitutive promoters. Copyright © 2010 John Wiley & Sons, Ltd.