The transcriptional activator ADR1, which is required for ADH2 and peroxisomal gene expression, contains four activation domains (TADs).We investigated the cofactors required by the TADs for activation of transcription. Mutations in ADA2 or GCN5, encoding components of the ADA2 coactivator complex, severely reduced LexA-ADR1-TAD activation of a LexA-lacZ reporter gene. Similarly, the ability of the wild-type ADR1 gene to activate an ADH2 driven promoter was compromised in strains deleted for ADA2 or GCN5.Using an in vitro protein binding assay, ADA2 and GCN5 were found to specifically contact individual TADs. ADA2 could bind TAD II, and GCN5 physically interacted with all four TADs. TAD IV deletion analysis indicated that its ability to bind GCN5 directly correlated with its ability to activate transcription in vivo. The ability of ADR1 TADs to contact TFIIB was also analyzed. Both TADs I and IV were shown to make specific contacts to TFIIB. These results suggest that ADR1 TADs make several contacts and may help explain the partial redundancy of ADR1 activation domains and their varying requirement at different promoters.The contact to and dependency on GCN5, a histone acetyl transferase, suggests that rearrangement of nucleosomes may be one important means by which ADR1 activates transciption. Mapping of GCN5 domains required for interaction with ADR1 TADs is currently in progress.