A human protein that is 92% identical and 97% homologous at the amino acid level to RanBP1 from mouse was identified by the two-hybrid method, using two types of target cDNAs fused to sequences encoding the GALA DNA-binding domain. The target cDNAs encoded the human Ran/TC4 and human RCC1 proteins, respectively. An in vitro binding experiment showed that RanBP1 binds to RCC1 with the aid of Ran. Partially purified, GST-fused RanBP1 inhibited RCC1-stimulated guanine nucleotide release from Ran in vitro. Consistent with this in vitro finding, overproduction of human RanBP1 was detrimental to growth of tsBN2, a temperature-sensitive BHK21 hamster cell line defective in the RCC1 gene, and inhibited the growth of the Saccharomyces cerevisiae reel mutants prp20, mtr1 and srm1. The specific effect of RanBP1 on rcc1(-) cells was confirmed by the finding that overproduction of RanBP1 induces significant levels of expression of a FUS1-lacZ gene and an increase in mating efficiencies in a ste3, pheromone receptor-deficient yeast mutant. This phenotype is similar to the srm1, a mutant isolated as a suppressor that restores mating to receptorless mutants. These findings indicate that RanBP1 negatively regulates RCC1.