The mouse mammary tumor virus (MMTV) promoter is transcriptionally silent prior to hormonal induction, partly because its organization into phased nucleosomes precludes access of transcription factors to their cognate sites. A T47D-derived cell line carrying a single integrated copy of the MMTV promoter exhibited a positioned nucleosome, which prevented binding of nuclear factor I (NFI). To study the molecular mechanisms controlling promoter accessibility we have made use of a strong chimeric transactivator, NFI-VP16, composed of NFI linked to the transactivation function of VP16. T47D cells transiently transfected with an MMTV-CAT reporter show little transcription even after cotransfection of an expression vector for NFI-VP16. However, a truncated MMTV promoter, lacking the hormone regulatory region (HRR) was transactivated by cotransfected NFI-VP16. The repressive effect of the HRR was not due to binding of a sequence-specific transcriptional repressor, and was evident with the DEAE-Dextran transfection procedure but not with the calcium phosphate technique. A similar behavior was observed in Saccharomyces cerevisiae carrying wild type or truncated MMTV-lacZ reporters and expressing NFI-VP16. Reconstitution experiments suggest that the promoter lacking the HHR generates less stable nucleosomes, a fraction of which contain a more accessible NFI site. Recombinant NFI binds to nucleosomes assembled on this truncated promoter but not to nucleosomes encompassing the HRR. These results are compatible with the notion that transiently transfected MMTV promoters behave like their stably integrated counterparts, in that the HRR drives positioning of a nucleosome and mediates transcriptional repression by preventing access of NFI to its cognate site.