MCM3 is an essential gene involved in the maintenance of minichromosomes in yeast cells. It encodes a protein of 971 ami no acids that shows striking homology to the Mcm2 protein. We have mapped the mcm3-1 mutation on the left arm of chromosome V approximately 3 kb centromere proximal of anpl. The mcm3-1 mutant was found to be thermosensitive for growth. Under permissive growth conditions, it was defective in minichromosome maintenance in an autonomously replicating sequence-specific manner and showed an increase in chromosome loss and recombination. Under nonpermissive conditions, mcm3-1 exhibited a cell cycle arrest phenotype, arresting at the large-bud stage with an undivided nucleus that had a DNA content of nearly 2n. These phenotypes are consistent with incomplete replication of the genome of the mcm3-1 mutant, possibly as a result of limited replication initiation at selective autonomously replicating sequences leading to cell cycle arrest before mitosis. The phenotype exhibited by the mcm3 mutant is very similar to that of mcm2, suggesting that the Mcm2 and Mcm3 proteins may play interacting roles in DNA replication.