A plaque-purified isolate of the Holland strain of Anatid herpesvirus (AHV-ppc3) was purified by differential and buoyant density sedimentation. The virus buoyant density was 1.215 g/cm³ in sucrose. AHV-ppc3 DNA was analyzed by sedimentation velocity studies in neutral or alkaline sucrose gradients. Based upon a comparison with T4 DNA, the DNA of AHV-ppc3 was found to have a sedimentation coefficient of 59.7 S and a molecular mass of 1.19 × 10⁸ daltons. Between 15 and 22 bands were observed in agarose gel electrophoresis with AHV DNA cut by BarnHl, EcoRl, Pstlor BgHIThe possibility of isometric forms is indicated by the finding of restriction fragments having molar ratios differing from 1.0. The mean molecular mass calculated from the fragments was 1.18 × 10⁸ daltons. Terminal fragments were identified using exonuclease II and BgHI. In alkaline sucrose gradients, AHV DNA fragments with the largest of the most abundant species have a sedimentation coefficient of 69 S and a calculated mass of 6.0 × 10⁷ daltons. The buoyant density of AHV-ppc3 DNA in cesium chloride was 1.723 g/cm³ corresponding to a % G + C content of 64.3. This finding was supported by thermal melts of the DNA in SSC/10 in which the thermal melting point was 82.7°. The data support a model for AHV in which the viral duplex DNA is linear, without covalently closed termini or significant base modifications, but with single strand nicks or gaps. The % G + C content of AHV DNA is the highest reported for any avian herpesvirus in the α-herpesvirinea subgroup.