Protein synthesis was determined in cultures of human umbilical cord vein endothelial cells (EC) infected with either herpes simplex type 1 (HSV-1) or type 2 (HSV-2). Monolayers were infected for 1 hour with either 5 or 20 infectious virus particles per EC (multiplicity of infection of 5 or 20). At different times after infection, infected and noninfected cultures were pulsed with either 5 mu Ci/ml of [14C]proline or 25 mu Ci/ml of [35S]methionine for 1 or 2 hours. Autoradiograms of sodium dodecyl sulfate-gel electrophoresis showed suppression of matrix protein synthesis by 4 hours postinfection which became almost complete at 6 hours. Multiplicity of infection of 20 was more effective than multiplicity of infection of 5 at suppressing matrix protein synthesis at 4 or 6 hours postinfection. Infection of EC with ultraviolet light-inactivated virus resulted in the shutoff of host-cell as well as virus protein synthesis. Similar results were observed when monolayers of EC were infected with intact virus in the presence of 2 micrograms/ml of actinomycin-D, an inhibitor of RNA synthesis. On the other hand, uninfected EC in the presence of actinomycin-D continued to synthesize protein from pre-existing mRNA. The time of shutoff of synthesis of specific matrix proteins varied with the protein i.e., shut-off of type IV collagen occurred first, followed by that of fibronectin, and then of thrombospondin. The data suggest that the degree of suppression of synthesis of EC matrix proteins after infection with HSV-1 or HSV-2 is dependent on the dose of the virus inoculum; it occurs at the translational level and is not dependent on new viral protein synthesis.