Ribonucleotide reductase comprises two nonidentical protein subunits R1 and R2, both of which are required for enzyme activity and show cell cycle-dependent regulation. The TATA-less promoter of the human RRM1 gene (encodes R1 protein) was examined with reference to regulatory domains upstream of the transcription start site. A region from nt -195 to +3 was found to give maximal expression of a reporter gene when transfected into the human cell line K562. Overall, this 198-bp region shows 58% identity with the equivalent region of the murine promoter; however, it contains two 22-bp domains that were 81 and 91% identical between species. Electrophoretic mobility shift assays were performed using a fragment of the domain closest to the transcription start site. These experiments revealed that several factors were able to bind this region in a sequence-specific manner. One of these factors was shown to be Sp1 by specific competition and supershift using antibody to Sp1. The data presented suggest that Sp1 is involved in the transcription of RRM1.