It has been shown previously that amiloride, a widely used diuretic drug, inhibits growth in Schizosaccharomyces pombe. Here we show that the drug also alleviates repression by various nutrients of mating and sporulation in fission yeast. We selected spontaneous mutants that are amiloride-resistant and unable to mate and sporulate. One of them defines the gene ste20. This gene has been cloned and sequenced. It codes for a putative protein of 1309 amino acids. Its sequence does not provide any clues to its function. In contrast to the wild-type, mutants defective in this gene can grow in a medium containing 40 microm amiloride, do not arrest in G(1), and do not induce ste11 expression upon nitrogen starvation and thus are sterile. In addition the ste20 mutants are methylamine-sensitive, exhibit enhanced medium acidification and are defective in the utilization of gycerol as a carbon source.