Progression through G2/M of the mammalian cell division cycle requires the coordinated expression of many gene products, but little is known of the transcriptional regulators involved. Schizosaccharomyces pombe Cdc5 is a putative transcription factor implicated in G2/M transit. We recently identified a cDNA encoding a putative human transcription factor, now designated human Cdc5 (hCdc5), with homology to S. pombe Cdc5. Widespread expression of hCdc5 in human tissues and homology with expressed sequences in other eukaryotes suggested an evolutionarily conserved general function. Nuclear import of hCdc5 upon serum stimulation of mammalian cells suggested a possible role in cell proliferation. We now report that overexpression of hCdc5 in mammalian cells shortened G2 and reduced cell size. A dominant negative mutant of hCdc5 lacking the carboxyl-terminal activation domain slowed G2 progression and delayed entry into mitosis. Thus, hCdc5 is the first transcriptional regulator shown to affect G2 progression and mitotic entry in mammalian cells.