Histone acetyltransferase (HAT) activity has been demonstrated for several transcriptional activators, formally connecting chromatin modification with gene regulation. However, no effect on chromatin has been demonstrated. We have investigated the role of the HAT Gcn5 at the nucleosomally regulated PHO5 promoter. Under conditions of constitutive submaximal activation (i.e., in the absence of the negative regulator Pho80), deletion of Gcn5 determines a novel randomized nucleosomal organization across the promoter and leads to a dramatic reduction in activity. Furthermore, mutation of amino acids critical for Gcn5 HAT activity is sufficient to generate this structure. This intermediate state in chromatin opening gives way to the fully open structure upon maximal induction (phosphate starvation), even in the absence of Gcn5. Thus, Gcn5 is shown to affect directly the remodeling of chromatin in vivo.