It has been suggested that a conserved heptad repeat region in paramyxovirus fusion (F) proteins is essential for viral fusion activity (Bucklandet al.,1992; Sergelet al.,1994; Reitteret al.,1995). We have studied synthetic peptides containing the heptad repeat regions derived from the F proteins of human parainfluenza virus type 2 (PI2) and type 3 (PI3) for their function as potential inhibitors of virus-induced cell fusion as well as their effects on spread of viral infection. Two peptides containing sequences of heptad repeat B, adjacent to the transmembrane domain of the F protein, were synthesized for both PI2 and PI3 F proteins. We observed that the longer peptides [34 amino acids (a.a.) for PI2F or 35 a.a. for PI3F] which extend from heptad repeat B to the transmembrane domain showed complete inhibition of cell fusion induced by the respective virus as well as by the vaccinia-expressed F and HN proteins. The 50% effective concentration to inhibit virus-induced cell fusion was 2.1 μMfor PI2 and 1.2 μMfor PI3. Moreover, the inhibitory effects of each peptide on virus-induced cell fusion were found to be virus type-specific. These peptides were found to also inhibit viral entry and to prevent plaque formation when mixed with the virus inoculum. Furthermore, the peptides caused a reduction in virus yield when assayed 48 hr after low m.o.i. infection and in the size of viral plaques when added to the overlay. Shorter peptides (21 a.a. for PI2F or 24 a.a. for PI3F) which correspond to the partial sequence of heptad repeat B for PI2F and the entire heptad repeat B for PI3F showed partial inhibition of PI2- or PI3-induced cell fusion. These results indicate that peptides containing the heptad repeat B sequence have the potential to inhibit virus-induced cell fusion, virus entry, and spread of virus infection.