The Schizosaccharomyces pombe mei3(+) gene is expressed only in diploid cells undergoing meiosis. Ectopic expression of mei3(+) in haploid cells causes meiotic catastrophe. Mei3 is an inhibitor of Ran1/Pat1 kinase and contains a nine-amino-acid motif, Mei3-RKDIII, that resembles two regions in the Ste11 substrate for Ran1/Pat1. Substitution of serine for Arg-81 within Mei3-RKDIII transforms the inhibitor into a substrate for Ran1/Pat1. Thus, it is likely that Mei3-RKDIII defines a pseudosubstrate sequence. In this study, we constructed a series of mei3 deletion mutations and assayed each for activity. This analysis indicates that the carboxy-terminal domain of Mei3 is sufficient for function in vivo. Alanine-scanning mutagenesis identifies critical residues within the inhibitory domain. Two mutations, SM1 and SM8, fail to cause meiotic catastrophe. The SM1 mutation contains alterations of amino acid residues in Mei3-RKDIII. Recombinant SM1 protein exhibits reduced ability to inhibit Ran1/Pat1 kinase in vitro and interacts inefficiently with the kinase in a two-hybrid assay. The SM8 protein binds to Ran1/Pat1 in a two-hybrid assay but fails to inhibit Ran1/Pat1 substrate phosphorylation in vitro. These findings provide evidence that Mei3-RKDIII defines a Ran1/Pat1-binding site that is necessary but not sufficient for inhibition of the kinase. Using fusions to green fluorescent protein, the cellular localization of Ran1 and Mei3 was examined in living cells. Ran1 is concentrated in the nucleus. Mei3 is also enriched in the nucleus and, consistent with the genetic and biochemical results, the inhibitory domain of Mei3 is sufficient for nuclear localization.