The effect of violamycin BI (VBI) was investigated in different cell substrates, infected or not with herpes simplex virus type 1 (HSV-1), strains VR3 and Rapp-1. The cytotoxic dose of VBI was established in calf kidney and human embryo cell cultures. Low drug concentrations induced moderate cytotoxic changes, allowing the study of the cells for 4-6 days. The action of VBI on HSV-1 was evident mostly in the early phases of the virus multiplication cycle. The antiviral effect of VBI was due both to the direct action of the drug on HSV and to its interaction with the nucleic acids of the host cells supporting HSV multiplication in their nuclei.