Mechanisms regulating the cell division cycle are well conserved among all eukaryotes. Consistently many proteins regulating the cell cycle are functionally interchangeable between many organisms. Cell division control is regulated on different levels of which the transcriptional level appears to be particularly important for controlling synthesis of many cell cycle proteins. We had earlier described transcription factor-binding sites essential for regulating genes important for the transition from the G(2) phase to mitosis. A tandem repressor site named cell cycle-dependent element (CDE) and cell cycle genes homology region (CHR) are responsible for the correct expression during the cell cycle. Another feature of these G(2)/M-specific promoters is the activation through 2 or 3 CCAAT boxes binding the transcription factor nuclear factor-Y (NF-Y). These major activating sites have to be spaced 32 or 33 bp apart to be fully functional. We were interested in looking at the evolutionary changes in regulatory elements and overall promoter structure of 3 well-characterized cell cycle genes. Here, we compare the DNA sequences and functional features of the cdc25C, cyclin B1, and cyclin B2 promoters from humans, mouse, chimpanzee, and orangutan. We find numerous differences in the nucleotide sequence between mouse and primate promoters. However, CHR and CCAAT boxes stand out in that they are perfectly conserved in all promoters tested. The CDE site contains nucleotide exchanges between mouse and primate promoters. Comparing sequences and functions of chimpanzee, orangutan, and human promoters, we observe a complete conservation in nucleotide sequence of the regulatory elements. Functional assays of the cyclin B1, cyclin B2, and cdc25C promoters yield moderate variations in activity and thereby a good conservation of function. Although we find nucleotide differences in cell cycle promoters between orangutan and humans of about 5%, there are never changes in any of the CCAAT boxes or CDE/CHR sites in the cyclin B1, cyclin B2, and cdc25C promoters. Furthermore, we describe the influence of the tumor suppressor p53 and the transcriptional activator NF-Y on regulation of the newly cloned primate promoters.