An aqueous extract from toad skin, cinobufacini, has been known to possess anticancer ability. The present study examined effect of toad skin extract on activity of gelatinases including matrix metalloproteinases-2 and -9 which play an important role in invasion of carcinoma cells. Gelatinase activities derived from fetal serum albumin and culture medium of human breast carcinoma cell line MDA-MB-231 were significantly prevented in the presence of toad skin extract. The inhibitory activity was found in water-soluble fraction of the extract prepared by the Bligh & Dyer method but not in CHCl(3)-soluble lipid fraction. These results suggest that an aqueous extract from toad skin contains a water-soluble substance possessing a potent ability to prevent gelatinase activity. In conclusion, the water-soluble substance in toad skin extract cinobufacini may be able to regulate cancer cell migration accelerated by matrix metalloproteinases.