Genetic experiments in the yeast Saccharomyces cerevisiae have identified the five Not proteins as global repressors of transcription which preferentially repress core promoters which do not contain a canonical TATA sequence. Recently, the Ccr4 and Caf1 proteins, required for non-fermentative gene expression, were found to be associated with the five Not proteins in 1.2 and 2 MDa Ccr4-Not complexes. These Ccr4-Not complexes, as many other global regulators of transcription, appear to regulate transcription both positively and negatively in vivo. To further characterize the activity of Not1p, the only essential known protein of the Ccr4-Not complex, and determine whether it can act directly as a transcriptional repressor, we established an in vitro transcription system in which the HIS3 TATA-less promoter can be efficiently transcribed. We demonstrate that transcription from the HIS3 TATA-less promoter can be specifically increased in vitro by preparing nuclear extracts from a conditional mutant of the NOT1 gene and analyzing transcription after shifting the nuclear extracts to the restrictive temperature. This result is the first demonstration that one of the Not proteins directly represses transcription. Moreover, it now defines an experimental system in which TATA-less transcription initiation and repression by the Ccr4-Not complex can be studied further.