Primers based on sequences flanking the vaccinia virus (VV) strain IHD hemagglutinin protein (HA) open reading frame (ORF) enabled amplification of HA DNA segments from the genome of raccoon poxvirus (RCN) and VV strain WR. The amplified segments produced unequal cross-hybridization signal intensities against each other, indicating sequence differences between the HA of RCN (in HindIII-G) and that of VV-WR (in HindIII-A). About 1.5 kb of sequences in the HA region were then determined from clones pRCN/HindIII-G and pVV/BamHI-32, a subclone of VV-WR HindIII-A. Pairwise analyses of the RCN and VV-WR HA nucleotide sequences showed 82, 66, and 86% homology, respectively, between the putative promoter, ORF, and transcript terminator regions and 53% homology between the deduced amino acid sequences of the HA of RCN (310 residues) and those of VV-WR (314 residues). The deduced HA amino acid sequences showed a putative signal peptide and transmembrane-anchor moiety of 70 and 62% homology and a rather distinct central domain (residues 146 to 254) of 32% homology. Additional hybridizations with the amplified segments described above enabled location of the HA gene in the HindIII-A fragment of the orthopoxviruses volepox virus (VPX) and skunk poxvirus (SKP); however, amplified DNA of either the entire HA ORF of RCN or that of VV-WR, or a portion, from the center to right end, did not hybridize with VPX or SKP, suggesting that the HA of RCN, VV, VPX, and SKP are rather diverged from each other. The VV HA was found to be closely related to that of ectromelia and variola viruses. The data are consistent with reports of hemagglutination-inhibition partial cross-reactivity between RCN, VPX, VV, and other orthopoxviruses and might lead to an explanation of the basis of syncytia formation by RCN, VPX, and SKP.