It has been suggested that a conserved heptad repeat region in paramyxovirus fusion (F) proteins is essential for viral fusion activity (Buckland et al., 1992; Sergel et al., 1994; Reitter et al., 1995) We have studied synthetic peptides containing the heptad repeat regions derived from the F proteins of human parainfluenza virus type 2 (Pl2) and type 3 (Pl3) 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 Pl2 and Pl3 F proteins. We observed that the longer peptides [34 amino acids (a.a.) for Pl2F or 35 a.a. for Pl3F] 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 microM for Pl2 and 1.2 microM for Pl3. 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 Pl2F or 24 a.a. for Pl3F) which correspond to the partial sequence of heptad repeat B for Pl2F and the entire heptad repeat B for Pl3F showed partial inhibition of Pl2- or Pl3-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.