Fission yeast flocculates nonsexually by induction of the flocculin encoded by gsf2(+) which is controlled by the positive regulator Mbx2. Here, we report a novel gene designated gsf1(+) found to be a negative regulator of nonsexual flocculation. We identified gsf1(+) as a multicopy suppressor of a sam2 mutation, which caused growth sensitivity to Ca(2+) and also found a nonsense mutation in gsf1(+) in a previously isolated gsf1 mutant. The gsf1(+) gene encodes a 547-aa protein containing a Zn(2)-Cys(6) binuclear cluster-type zinc finger motif. The Gsf1 protein localized in the nucleus, consistent with a role as a transcription factor. Deletion of gsf1(+) resulted in nonsexual flocculation inducible by CaCl2 , which was suppressed by the addition of EDTA or galactose. Both gsf2(+) and mbx2(+) were highly expressed in the gsf1 mutant. gsf1∆ gsf2∆ and gsf1∆ mbx2∆ double mutants did not flocculate, suggesting that gsf1(+) is an upstream regulator. In addition, the gsf1 mutant was sensitive to CaCl2 , KCl, HU, and TBZ, consistent with the possibility that gsf1(+) plays a role in functions unrelated to flocculation. Taken together, these results suggest that nonsexual flocculation in fission yeast is negatively controlled by Gsf1, which controls expression of mbx2(+) and gsf2(+) .