The spindle assembly checkpoint modulates the timing of anaphase initiation in mitotic cells containing improperly aligned chromosomes and increases the probability of successful delivery of a euploid chromosome set to each daughter cell. We have characterized cDNA sequences from several organisms with highly significant predicted protein sequence homologies to Saccharomyces cerevisiae Bub1p, a protein required for function of the spindle assembly checkpoint in budding yeast. The localization of mouse and human orthologs is in agreement with known conservation of synteny. Mouse backcross mapping data indicate that the murine gene resides on chromosome 2 near IL1A, 73 cM from the mouse centromere. Radiation hybrid mapping data indicate that the human locus exhibits linkage to microsatellite marker D2S176, which is located within 10 cM of human IL1A. Multiple-tissue Northern analysis indicates conservation of expression pattern in mouse and human with markedly high mRNA levels in testis. Northern analysis of two different spindle assembly checkpoint protein gene products from human, BUB1 and MAD2, reveals an expression pattern with common tissue distribution consistent with roles in a common pathway. In addition, we demonstrate that an mRNA found to accumulate in a rat fibroblast cell transformation system encodes rat BUB1, and we find that rat BUB1 mRNA accumulation correlates with the proliferation status of cells in culture.