A number of polysaccharides showed good antiviral activity against several animal viruses. At 5 micrograms/ml, carrageenan prevented the cell monolayer from destruction by herpes simplex virus type 1 (HSV-1) growth. At 10 micrograms/ml, carrageenan reduced the formation of new infectious HSV-1 by almost five logs. No cytotoxic effects were detected with concentrations of carrageenan up to 200 micrograms/ml. When 10 micrograms of carrageenan per ml was added at the beginning of HSV-1 infection of HeLa cells, there was potent inhibition of viral protein synthesis, and the cells continued synthesizing cellular proteins. This did not occur if carrageenan was added 1 h after HSV-1 infection. The use of [35S]methionine-labeled virions to analyze the entry of HSV-1 or Semliki Forest virions into cells indicated that carrageenan had no effect on virus attachment or virus entry. Moreover, carrageenan did not block the early permeabilization of cells to the toxic protein alpha-sarcin. These results suggest that this sulfated polysaccharide inhibits a step in virus replication subsequent to viral internalization but prior to the onset of late viral protein synthesis.