Human cytomegalovirus (HCMV) is released into plasma during active infection. To characterize cell-free virus interaction with blood components, the association of HCMV with washed platelets was assessed in binding assays. HCMV binding was regulated by divalent cations and inhibited in the presence of anti-HCMV antibody and excess unlabeled virus by 80% and 66%, respectively. Addition of plasma caused a 2.5-fold increase in binding to platelets. HCMV-induced platelet aggregation occurred more efficiently in plasma (14% +/- 4% in 5 min) than in buffer (8% +/- 2% in 15 min). Plasma fibronectin bound HCMV and increased the extent of HCMV-mediated aggregation of washed platelets by > 2-fold. Plaque assays indicated that platelet-associated HCMV retained its capacity to infect fibroblasts. Plasma and fibronectin inhibited viral infectivity by 81% and 70%, respectively. Thus, platelets and plasma components may play an intermediary role in HCMV infection and dissemination.