Clinical isolates of human parainfluenza virus type 1 in our laboratory were found to induce significantly different degrees of syncytium formation in CV-1 cells. Sequence analysis of high- and low-fusion strains suggested that the hemagglutinin-neuraminidase (HN) protein was responsible for the differences in fusion activity. We exploited the strain differences to define the specific amino acid residues of the HN protein which were responsible for the low and high fusion activities. The HN proteins of the two low-fusogenic strains 8389 and 45785, and the highly fusogenic strain C35, were expressed in HeLa T4+ cells and their fusion promotion activities were compared. When coexpressed with C35 F, HNs from the low-fusogenic viruses were associated with much lower fusion activity than was C35 HN, suggesting that the HN proteins modified the fusogenicity of the viruses. To identify the region of the HN protein responsible for this difference, we constructed a series of chimeric HN cDNAs combining 8389 and C35 sequences. All chimeric HNs that contained C35 sequence in the central 36% of the protein exhibited high fusion promotion activity. Further analysis by site-directed mutagenesis showed that a single Asn-to-Lys substitution at position 242 converted 8389 HN to a highly fusion-promoting molecule. Thus, the globular head of the HN molecule is involved in fusion promotion activity.