The sequences involved in the regulation of transcription of the yeast ASP3 gene have been studied by the Bal31 deletion analysis. A plasmid YKW3 carrying the entire ASP3 gene and its upstream sequences was used to generate a series of deleted subclones. Deletion of a region between YKW3-6 (~-450) and YKW3-7 (~-300) abolished nitrogen catabolite repression (NCR) sensitivity, but still contain asparaginase II activity. However, when a region between YKW3-7 (~-300) and YKW3-8 (~-200) was deleted, it caused a complete absence of both NCR sensitivity and asparaginase II activity. These results indicate that a region between subclone YKW3-6 and YKW3-7 is essential for the transcriptional repression of the ASP3 gene expression under NCR and a region between subclone YKW3-7 and YKW3-8 is directly related to the transcriptional activation of the ASP3 gene.