We have cloned and characterized the MCM16 gene required for the maintenance of minichromosomes in the yeast Saccharomyces cerevisiae. This gene corresponds to a 181-amino acid ORF, YPR046W, on chromosome XVI. Mutant cells carrying minichromosomes accumulate them in higher copy numbers than do wild-type cells. Intact dicentric plasmid could be recovered from the mutant, in contrast to the wild-type, in which the plasmid suffered frequent deletions. A wild-type centromere, CEN6, acts as a block to the transcription of a reporter gene, such as beta-galactosidase. This block was less effective in the mutant than in the wild-type strain, suggesting alterations in kinetochore assembly in the former. The mutant also showed increased sensitivity to the antimitotic drugs benomyl and thiabendazole. The mcm16 mutation caused a high rate of loss of chromosome III, without any significant increase in the recombination frequency. A strain carrying a deletion-disruption derivative of the MCM16 gene was viable and, when compared to the wild-type, did not show any significant changes in growth rate or cell morphology at 16, 23 and 37 degrees C. These properties show that MCM16 is required for an important but nonessential role that governs the kinetochore-microtubule mediated process of chromosome segregation.