Pulmonary surfactant protein A (SP-A) has been shown to act as an opsonin in the phagocytosis of viruses by alveolar macrophages. To determine whether SP-A binds to viral proteins and which part of the SP-A molecule is involved in this interaction, binding studies were undertaken. SP-A was labeled with fluorescein isothiocyanate, and its binding to herpes simplex virus type 1-infected HEp-2 cells, as a model for virus-infected cells in general, was studied using flow cytometry. The binding of SP-A to virus-infected cells was saturable, reversible, and both time- and concentration-dependent, reaching a maximal level after 30 min at an SP-A concentration of 10 micrograms/ml. An approximately 4-fold increase in binding of SP-A to infected cells over control cells was observed. Yeast mannan, a mannose homopolysaccharide, did not influence the binding. However, heparin inhibited binding of SP-A in a concentration-dependent manner. In addition, heparin could also dissociate cell-bound SP-A, indicating that polyanionic oligosaccharides are involved in the binding of SP-A to virus-infected cells. Deglycosylated SP-A, obtained by digestion with N-glycosidase F, did not bind to infected cells. Heparin or deglycosylation of SP-A had no effect on the stimulation of alveolar macrophages by SP-A. It is concluded that the carbohydrate moiety of SP-A is involved in the recognition of viruses by SP-A and may play a role in the antiviral defenses of the lung.