The antiviral properties of neonatal and adult human neutrophils were investigated by their ability to inhibit the formation of herpes simplex virus (HSV) plaques using an extrinsic viral resistance (EVR) assay. The EVR assay was performed by incubating neutrophils or mononuclear cells (MNC) with HSV-infected Vero or CEM tumor cells for 48 h. The cells were then frozen and viable HSV was quantitated by the ability of the lysate to form viral plaques. Activation of neutrophils from normal adults with phorbol myristate acetate increased their ability to inhibit HSV plaque formation more than 10-fold. The EVR response of neutrophils from newborn infants was much lower, and no significant inhibition occurred using activated neutrophils from patients with chronic granulomatous disease. The presence of rabbit anti-HSV antiserum slightly increased the EVR response of neutrophils but produced a greater increase in the response of the MNC in both adults and newborns. However, the combination of antiserum plus cytokines (granulocyte-macrophage colony-stimulating factor, IL-2, and interferon-gamma) greatly augmented the neutrophil EVR response to the level of the MNC response. Thus, neutrophils are capable of exerting a strong antiviral response comparable to that of MNC that may be important as a second line of defense in the immunocompromised patient.