Enhancers are generally viewed simply as extensions of promoters, lacking a function of their own. However, previous studies of mouse preimplantation embryos revealed that 1-cell embryos can utilize enhancer-responsive promoters efficiently without an enhancer, whereas 2-cell embryos require an enhancer to achieve the same levels of expression. This suggested that enhancers relieved a repression in 2-cell embryos that is absent in 1-cell embryos. Results presented here demonstrate first that the ability of 1-cell embryos to dispense with enhancers does not result from the absence of specific activation proteins. Under conditions where GAL4-VP16 activated a GAL4-dependent promoter in both embryos, GAL4-VP16 activated a GAL4-dependent enhancer only in 2-cell embryos. Moreover, the role of an enhancer is not to compensate for either changes in promoter requirements, or for reduced levels of promoter-specific transcription factors. Linker-scanning mutations in a natural promoter revealed that both embryos utilized the same promoter elements, and comparison of different promoters revealed that these embryos have equivalent transcriptional capacities. In addition, titration experiments revealed less Sp1 activity in 1-cell embryos where enhancers are dispensable than in 2-cell embryos where enhancers are required. Therefore, we propose that the primary function of enhancers, first evident with formation of a mouse 2-cell embryo, is to prevent repression of weak promoters, probably by altering chromatin structure. Consistent with this hypothesis is the fact that butyrate, an agent that alters chromatin structure, stimulated promoters in 2-cell embryos, but not in 1-cell embryos.