The RNR3 gene encodes the large subunit of ribonucleotide reductase. Transcription of this gene is induced 12-fold in response to DNA damage or by a DNA replication blocker. To investigate cis-acting regulation, deletion analysis of the promoter region of the RNR3 gene was performed and we identified two upstream-repressing sequences in the RNR3 regulatory region. An 18-base-pairs fragment, termed DNA-damage responsive element 1 (DRE1) located between -212 and -194 in this region was found to be essential for the induction of RNR3. This fragment contained a negatively acting sequence where a protein factor bound to the region during normal growth but disappeared by exposure to 4-nitroquinoline-1-oxide. The other repressive element homologue to DRE1 was located at -263 to -254. One possible upstream-activating sequence which regulates the basal expression of RNR3 was also found. These results show that at least three potential cis-elements are necessary for the inducible expression of yeast expression of yeast RNR3 in response to DNA damage.