Recently, cDNA corresponding to the human homologue of the BUB3 (budding uninhibited by benomyl) mitotic checkpoint protein has been identified and cloned. Previous studies from our laboratory and others have found this gene to localize to 10q26, a region that is frequently altered in various human cancers. We describe here a series of studies designed to understand the genomic structure of BUB3, particularly as it relates to regulation of gene expression. The human BUB3 gene has seven exons and six introns, and spans a genomic region of over 16 kb. The four WD repeat sequences in this gene are localized to exons 2, 4, and 6, and there is a major transcriptional start site located 554 nucleotides upstream of the ATG translation initiator codon. The promoter region lacks a TATA box but contains potential binding sites for the transcriptional factors including SP1, E2F, c-Myc, C/EBP and NFkappaB. To analyse the regulatory mechanisms controlling hBUB3 gene expression, we characterized the 5'-flanking region from nucleotide -1.3 to +0.58 kb by cloning various potions of this region in front of a luciferase reporter sequence. These experiments indicate that this region 5' region contains distinctive positive and negative regulatory elements.