Nucleoticidin and melanocidins A and B exhibited potent inhibitory activity against 5'-nucleotidases from rat liver membrane and snake venom. These inhibitors are polysaccharides with highly branched side chains having at least disaccharide units. This conclusion was supported by the results with polysaccharides of known chemical structures. The inhibitors showed non-competitive inhibition with respect to AMP, and urea-treatment caused a marked decrease or a disappearance of the 5'-nucleotidase inhibitory activity. Therefore, it is concluded that steric factors also play an important role in their inhibitory activity.