Like many solid tumours, oral squamous cell carcinomas (OSCC) invariably exhibit chromosomal instability (CIN) leading to aneuploidy. The mechanisms responsible for CIN in OSCC, however, are largely unknown. This study examined the fidelity of the spindle checkpoint, together with the number, structure and function of centrosomes in a series of well-characterised aneuploid immortal OSCC-derived cell lines that harbour p53 and p16(INK4A) defects. The spindle checkpoints were fully functional in 2 of 7 cell lines and attenuated in the remaining 5 cell lines. Overexpression of the spindle checkpoint protein, Cdc20, was observed in 2 of the cell lines with attenuated checkpoints. Defects in centrosome number, size and localisation were detected in 5 of the cell lines. Clonal cell populations contained cells with both normal and abnormal numbers of centrosomes, suggesting that the control of centrosome number may be inherently unstable in OSCC-derived cell lines. Centrosomal abnormalities were then examined in tissue samples of oral epithelial dysplasias and carcinomas. Abnormal centrosomes were detected in all the tissues examined albeit in a low percentage of cells (<1% to >5%). The percentage of cells containing centrosome abnormalities was significantly higher in the carcinomas than in the dysplasias (p<0.02) and in the poorly differentiated SCCs relative to their moderately differentiated (p<0.04) and well-differentiated (p<0.01) counterparts. We suggest that the genetic alterations associated with the development of the immortal phenotype, together with spindle checkpoint and centrosome defects, are responsible, albeit in part, for the complex karyotypes observed in OSCC. The presence of centrosome abnormalities in oral dysplasias raises the possibility that such defects might contribute to malignant progression.