The Saccharomyces cerevisiae NHP6A and NHP6B proteins are chromatin architectural factors, functionally and structurally related to the mammalian high mobility group (HMG)-1 and -2 proteins, a family of non-sequence-specific DNA binding proteins. nhp6a nhp6b mutants have various morphological defects and are defective in the induced expression of several RNA polymerase II-transcribed genes. We found that NHP6A/B proteins are also required for full induction of the yeast CHA1 gene. Importantly, CHA1 basal level expression is increased 10-fold in an nhp6a nhp6b double deletion mutant. Micrococcal nuclease and DNase I analysis of the CHA1 gene in this strain showed an open promoter structure, characteristic of the activated state of this promoter, even under non-inducing conditions. To address the possible function of the NHP6A/B proteins in chromatin-mediated gene regulation, we performed whole-genome transcriptional profiling of a Deltanhp6a Deltanhp6b yeast strain. Our results suggest that NHP6A/B proteins play an important regulatory role, repressing as well as potentiating expression of genes involved in several cellular processes, and that NHP6A/B control is exerted at the level of the individual gene.