CDC2 has been shown to regulate entry into mitosis in eukaryotic cells. However, in Aspergillus nidulans, activation of CDC2 itself is not sufficient to trigger mitosis if another mitotic protein kinase, NIMA, is not activated. Superficially, NIMA and CDC2 have analogous functions and are regulated in a similar manner. NIMA activity is tightly regulated during the cell cycle. Overexpression of NIMA induces germinal vesicle breakdown in Xenopus oocytes and promotes premature entry into mitosis in all eukaryotic cells examined, whereas dominant-negative mutant NIMA causes a specific G2 arrest in Aspergillus nidulans and human cells, as is the case for CDC2. However, NIMA and CDC2 have quite distinct primary sequence substrate specificities. Furthermore, the regulatory mechanisms that govern the cell cycle-dependent abundance, activity and localization are largely intramolecular for NIMA but intermolecular for CDC2. More importantly, a NIMA-like pathway is also required for the G2/M transition in vertebrate cells. Thus, NIMA may represent a new essential eukaryotic cell cycle regulator, although its homologues in other species are yet to be identified.