Mutations in Aurora of Drosophila and related Saccharomyces cerevisiae Ipl1 kinase are known to cause abnormal chromosome segregation. We have isolated a cDNA encoding a novel human protein kinase of 402 amino acids with a predicted molecular mass of 45.9 kDa, which shares high amino acid identities with the Aurora/Ipl1 protein kinase family; hence the cDNA is designated as aik (aurora/IPL1-related kinase). Amino acid sequence of C-terminal kinase domain of Aik shares 86, 86, 72, 59, and 49% identity with those of Xenopus XLP46APK and XLP46BPK, mouse STK-1, Aurora of Drosophila, and yeast Ipl1, respectively, whereas N-terminal domain of Aik shares high homology only with those of XLP46APK and XLP46BPK. Northern and Western blotting analyses revealed that Aik is expressed highly in testis and various proliferating cells including HeLa cells. In HeLa cells, the endogenous levels of aik mRNA and protein contents are tightly regulated during cell cycle progression. Both of these levels are low in G1/S, accumulate during G2/M, and reduce rapidly after mitosis. Its protein kinase activity is also enhanced at mitosis as inferred by exogenous casein phosphorylation. Immunofluorescence studies using a specific antibody have shown that Aik is localized to the spindle pole during mitosis, especially from prophase through anaphase. These results strongly suggest that Aik is a novel member of a protein kinase family possibly involved in a centrosome function(s) such as chromosome segregation or spindle formation.