The sulfated exopolysaccharide p-KG03, which is produced by the marine microalga Gyrodinium impudicum strain KG03, exhibited impressive antiviral activity in vitro (EC50 = 26.9 micro g/ml) against the encephalomyocarditis virus (EMCV). Depending on the p-KG03 concentration, the development of cytopathic effects in EMCV-infected HeLa cells was either inhibited completely or slowed. Moreover, p-KG03 did not show any cytotoxic effects on HeLa cells, even at concentrations up to 1000 microg/ml. The polysaccharide was purified by repeated precipitation in ethanol, followed by gel filtration. The p-KG03 polysaccharide had a molecular weight of 1.87 x 10(7), and was characterized as a homopolysaccharide of galactose with uronic acid (2.96% wt/wt) and sulfate groups (10.32% wt/wt). The biological activities of p-KG03 suggest that sulfated metabolites from marine organisms are a rich source of antiviral agents. This is the first reported marine source of antiviral sulfated polysaccharides against EMCV. The p-KG03 polysaccharide may be useful in the development of marine bioactive exopolysaccharide for biotechnological and pharmaceutical products.