The cation-dependent galactose-specific flocculation activity of the  Schizosaccharomyces pombe  null mutant of  lkh1   + , the gene encoding LAMMER kinase homolog, has previously been reported by our group. Here, we show that disruption of  prk1   + , another flocculation associated regulatory kinase encoding gene, also resulted in cation-dependent galactose-specific flocculation. Deletion of  prk1  increased the flocculation phenotype of the  lkh1   +  null mutant and its overexpression reversed the flocculation of cells caused by  lkh1  deletion. Transcript levels of  prk1   +  were also decreased by  lkh1   +  deletion. Cumulatively, these results indicate that Lkh1 is one of the negative regulators acting upstream of Prk1, regulating non-sexual flocculation in fission yeast.