We investigated the function of the histone H3/H4 chaperones anti-silencing function 1 (Asf1p) and chromatin assembly factor 1 (CAF-1) in global transcriptional regulation in budding yeast. Deletion of ASF1 or CAF-1 components led to global transcriptional misregulation, both activation and repression, of genes scattered throughout the 16 yeast chromosomes. To investigate direct effects on gene regulation, we developed an approach to destabilize Asf1p that results in its rapid degradation within minutes of transcriptional repression. Upon degradation of Asf1p, rapid global changes in gene expression occur without the requirement for passage through S phase or de novo protein synthesis. In particular, we demonstrate that the previously reported influence of Asf1p on histone gene expression is not a direct effect of loss of Asf1p. These data indicate that the histone chaperones CAF-1 and Asf1p regulate the gene expression of a broad array of genes in yeast and, in the case of Asf1p, this is likely to be due to a direct role in chromatin modulation during transcriptional regulation.