Abstract: The multi-functional histone variant Htz1 (S. cerevisiae H2A.Z) is acetylated on up to four N-terminal lysines: K3, K8, K10 and K14. It has thus been posited that specific acetylated forms of the histone could regulate distinct roles. Antibodies against Htz1-K8Ac, K10Ac, and K14Ac show that all three modifications are added by Esa1 acetyltransferase and removed by Hda1 deacetylase. Completely unacetylatable htz1 alleles exhibit widespread interactions in genome-scale genetic screening. However singly-mutated (e.g. htz1-K8R) or singly-acetylable (e.g. the triple mutant htz1-K3R / K10R / K14R) alleles show no significant defects in these analyses. This suggests that the N-terminal acetylations on Htz1 are internally redundant. Further supporting this proposal, each acetylation decays with similar kinetics when Htz1 transcription is repressed, and global proteomic screening did not find a single condition in which one Htz1Ac was differentially regulated. However while the individual acetylations on Htz1 may be redundant, they are not dispensable. Completely unacetylatable htz1 alleles display genetic interactions and phenotypes in common with and distinct from htz1Delta. In addition each Htz1 N-terminal lysine is deacetylated by Hda1 in response to benomyl and reacetylated when this agent is removed. Such active regulation suggests that acetylation plays a significant role in Htz1 function.