How chromatin-mediated transcription regulates the beginning of mammalian development is currently unknown. Factors responsible for promoter repression and enhancer-mediated relief of this repression are not present in the paternal pronuclei of one-cell mouse embryos but are present in the zygotic nuclei of two-cell embryos. Here we show that coinjection of purified histones and a plasmid-encoded reporter gene into the paternal pronuclei of one-cell embryos at a specific histone-DNA concentration could recreate the behavior observed in two-cell embryos: acquisition of promoter repression and subsequent relief of this repression either by functional enhancers or by histone deacetylase inhibitors. Furthermore, the extent of enhancer-mediated stimulation in one-cell embryos depended on the acetylation status of the injected histones, on the treatment of embryos with a histone deacetylase inhibitor, and on the developmentally regulated appearance of enhancer-specific coactivator activity. The coinjected plasmids in one-cell embryos also exhibited chromatin assembly, as determined by a supercoiling assay. Thus, injection of histones into one-cell embryos faithfully reproduced the chromatin-mediated transcription observed in two-cell embryos. These results suggest that the need for enhancers to stimulate promoters through relief of chromatin-mediated repression occurs once the parental genomes are organized into chromatin. Furthermore, we present a model mammalian system in which the role of individual histones, and particular domains within the histones that are targeted in enhancer function, can be examined using purified mutant histones.