An extract of culture medium of Lentinus edodes mycelia, JLS-S001, significantly blocked the release of infectious herpes simplex virus type 1 (HSV-1) from African green monkey kidney cells. The block in replication was not due to the effect of JLS-S001 on the adsorption and penetration of HSV-1 to the monkey kidney cells. This observation was supported by the fact that JLS-S001 had no significant effect on the expression of virus-specific nucleocapsid proteins in the treated cells. Furthermore, electron microscopy demonstrated the presence of nucleocapsids within the nuclei of the infected and JLS-S001-treated cells. However, the expression of glycoproteins B, C, D, E and I was reduced in the JLS-S001-treated cells. These results suggested that JLS-S001 blocked HSV-1 replication at a late stage in virus replication cycle probably in the assembly and budding of nucleocapsids and subsequent egress from the treated cells. © 1993.