Abstract: Eukaryotic cells have evolved a mechanism that delays the onset of anaphase until chromosomes are properly positioned on the spindle. To understand the molecular basis of such surveillance mechanism in human cells, we have cloned a full-length cDNA encoding a putative mitotic checkpoint kinase termed hBub1. Sequence comparison reveals that hBub1 is a structurally conserved protein, sharing 23% amino acid residue identity with BUB1 of budding yeast. In addition, the N-terminal portion (161 amino acids) of hBub1 shows a significant homology to yeast MAD3, a protein also known to be involved in the mitotic checkpoint response pathway. Northern blot analyses show that hBub1 mRNA level is abundantly expressed in tissues or cells with a high mitotic index. When Dami cells undergoes terminal differentiation following treatment with phorbol ester, hBub1 expression in this cell line is rapidly downregulated. The hBub1 protein level is low in G1 and remains relatively constant in S, G2 and M phases.