Polymorphonuclear leukocytes (PMN) can mediate cytotoxic reactions against virus infected targets cells. We observed very efficient binding of PMN to HSV-infected fibroblasts when loaded with HSV-specific antibodies. Using electron microscopy, infected fibroblasts were found to be totally surrounded by PMN and the phagocytosis of virions and fragments of infected cells was demonstrated. To quantify and study this phenomenon, and to compare PMN with monocytes, we developed radiometric and fluorometric phagocytosis assays. Leukocytes were mixed with [3H]glucosamine- or FITC-labeled virus and incubated at 37 degrees C. PMN associated radioactivity or fluorescence per cell as measured by flow cytometry was determined. PMN phagocytosis was dependent on the presence of specific anti-HSV antibodies and could be enhanced by addition of complement. Monocytes were also able to phagocytize virions; however, the rate of uptake was less than that for PMN. Under optimal conditions the total amount of herpes simplex particles that could be associated with one PMN or monocyte was about 10,000. PMN and monocytes are capable of phagocytosis of HSV. This may be an important factor in preventing the spread of infection in vivo.