We have performed a screen for genes that affect chromosome stability when overexpressed in the budding yeast Saccharomyces cerevisiae. Two of the genes recovered in the screen, CST17 and CST20, share a number of phenotypic properties, suggesting their involvement in the same cellular process. DNA sequence analysis of these genes revealed that they encode components of the Ran/RCC1 molecular switch system: CST17 is Ran itself (Ras-like nuclear protein) and CST20 is a novel yeast protein with a high degree of similarity to mammalian RanBP1, which is known to interact with Ran-GTP in vitro. We demonstrate that the CST20 protein can interact with Ran-GTP in vitro under similar conditions, indicating that it is the functional yeast homolog of mammalian RanBP1. The results of immunoprecipitation experiments show that the two yeast proteins form a complex in vivo. Deletion of the gene encoding RanBP1 revealed that it is essential for viability, as are Ran and RCC1. Similar phenotypic consequences of overproduction of either Ran or RanBP1 indicate that the latter protein is a functional component of the Ran/RCC1 molecular switch system, which is implicated in the control of a number of nuclear functions. Our finding that overproduction of two components of this system results in mitotic chromosome nondisjunction and sensitivity to an anti-microtubule drug benomyl suggest their involvement in mitosis as well. Thus RanBP1 is a functional component of a highly conserved molecular system that affects diverse cellular processes. The availability of this gene in S. cerevisiae provides a genetic system for the analysis of RanBP1 function in vivo.