Nucleosome loss from a promoter region has recently been described as a potential mechanism for transcriptional regulation. We investigated whether H3/H4 histone chaperones mediate the loss of nucleosomes from the promoter of the yeast PHO5 gene during transcriptional activation. We found that antisilencing function 1 (Asf1p) mediates nucleosome disassembly from the PHO5 promoter in vivo. We show that nucleosome disassembly also occurs at a second promoter, that of the PHO8 gene, during activation, and we demonstrate that this is also mediated by Asf1p. Furthermore, we show that nucleosome disassembly is essential for PHO5 and PHO8 activation. Contrary to the current dogma, we demonstrate that nucleosome disassembly is not required to enable binding of the Pho4p activator to its PHO5 UASp2 site in vivo. Finally, we show that nucleosomes are reassembled over the PHO5 promoter during repression. As such, nucleosome disassembly and reassembly are important mechanisms for transcriptional activation and repression, respectively.