Cyclin B1 mRNA expression varies through the cell cycle with its peak in G2/M. In cycling mammalian cells, its lowest level is in G1 with a steady increase in S until a level 50-fold greater than that in G1 is reached. In order to characterize the transcriptional component to this variation in expression, we cloned the upstream region 872 base pairs upstream from the start site of the cyclin B1 gene and have demonstrated that it confers cell cycle-dependent regulation onto two reporter genes, both chloramphenicol acetyltransferase and luciferase. Its activity was 25-fold greater in G2/M than in G1 in HeLa cells with intermediate activity in S. This cyclical activity could be seen with sequences encompassing only 90 base pairs upstream from the start site. Protein binding to this region was demonstrated using electrophoretic mobility shift assays, and the binding profiles appeared to vary depending upon the phase of the cycle in which the extracts are made. Thus, transcriptional control plays an important role in determining cyclin B1 mRNA levels, and cell cycle-dependent activity is regulated through interactions with the region 90 bases upstream from the start site.