Our current studies have demonstrated that human parainfluenza virus type 3 (HPIV-3) utilizes heparan sulfate (HS) for its efficient cellular entry. HPIV-3 interacted with HS-agarose in vitro and the cellular entry and infection of HPIV-3 were reduced following (a) infection of human epithelial lung A549 cells with HPIV-3 pre-incubated with soluble HS; (b) treatment of A549 cells with heparinase to remove cell surface HS and sodium chlorate (NaClO(3)), a potent inhibitor of proteoglycan sulfation; and (c) infection of HS-deficient mutant CHO cell lines. However, in each instance, complete inhibition of HPIV-3 entry did not occur, suggesting the presence of additional nonproteoglycan cell surface molecule(s) that is required for HPIV-3 entry. Thus the cell surface HS appears to play an important role in efficient cellular entry of HPIV-3.