The serine/threonine protein kinase NIMA of Aspergillus nidulans is required for entry into mitosis and may function in parallel to the universal mitotic inducer p34cdc2. Here, we report the isolation of complementary DNAs encoding Nek2 and Nek3, two novel human protein kinases structurally related to NIMA. Sequence comparisons revealed several unique features which may define a family of NIMA-related protein kinases. Nek2 was chosen for further study since it represents the closest known mammalian relative of NIMA. Chromosomal mapping of the nek2 gene identified two independent loci on chromosomes 1 and 14, and Northern blot analyses revealed the expression of two distinct mRNAs of approximately 2.4 and 4.7 kilobases in all human cell lines examined. In HeLa cells synchronized by both drug arrest and elutriation, a strikingly cell cycle-dependent pattern of Nek2 expression could be observed; Nek2 protein was almost undetectable during G1 but accumulated progressively throughout S, reaching maximal levels in late G2. These observations demonstrate that Nek2 resembles Aspergillus NIMA, not only in its catalytic domain, but also in its cell cycle-dependent expression. Hence, the human Nek2 protein kinase may also function at the onset of mitosis.