The involvement of chromatin structure and organization in transcriptional regulatory pathways has become evident. One unsolved question concerns the molecular mechanisms of chromatin remodeling during in vivo promoter activation. By using a high resolution in vivo analysis we show that when yeast cells are exposed to a regulatory signal the positions of specific nucleosomes change. The system analyzed consists of the basic elements of the Saccharomyces cerevisiae ADH2 promoter, whose two nucleosomes are shown to change the distribution of their positions by few nucleotides in the direction of transcription, when the glucose content of the medium is lowered. Such repositioning does not occur in the absence of the ADH2 transcriptional activator Adr1 or in the presence of its DNA-binding domain alone. A construct consisting of the DNA-binding domain plus a 43 amino acids peptide containing the Adr1 activation domain is sufficient to induce the same effect of the full length protein. Nucleosome repositioning occurs even when the catalytic activity of the RNA polymerase II is impaired, suggesting that the Adr1 activation domain mediates the recruitment of some factor in order to correctly preset the relevant sequences for the subsequent transcription steps.