A conditional centromere was constructed in Saccharomyces cerevisiae by placing the centromere of chromosome III immediately downstream from the inducible GAL1 promoter from S. cerevisiae. By utilizing growth conditions that favor either transcriptional induction (galactose-carbon source) or repression (glucose-carbon source) from the GAL1 promoter, centromere function can be switched off or on, respectively. With the conditional centromere we were able to radically alter the mitotic transmission pattern of both monocentric and dicentric plasmids. Moreover, it was possible to selectively induce the loss of a single chromosome from a mitotically dividing population of cells. We observed that the induction of chromosome III aneuploidy resulted in a dramatic change in cell morphology. The construction of a conditional centromere represents a novel way to create conditional mutations of cis-acting DNA elements and will be useful for further analysis of this important stabilizing element.