The yeast HIS3 gene has two core promoters: TC, a TATA-less element and TR, a canonical TATA element. Four genes encode global negative regulators of transcription that preferentially repress TC-dependent transcription: NOT1 (CDC39), NOT2 (CDC36), NOT3 and NOT4 (SIG1, MOT2). Genetic and biochemical experiments suggest that the products of these genes are associated in a complex and regulate TFIID function. In this paper, we describe a new gene, NOT5, that also represses transcription of the HIS3 TATA-less promoter preferentially and encodes a protein whose N-terminal region is 44% identical to that of Not3p. Our results indicate that NOT5 is involved in Not function and encodes a product that is physically associated with the other Not proteins. First, overexpression of NOT3 or NOT4 suppresses mutations in NOT5. Secondly, mutations in NOT4 are synthetically lethal with mutations in NOT5. Thirdly, NOT5 interacts with NOT1 and NOT3 in the two-hybrid assay. Finally, Not1p, Not3p and Not4p co-immunoprecipitate with Not5p.