Equilibrium centrifugation of entomopoxvirus DNAs from Lepidoptera (Amsacta moorei, Euxoa auxiliaris), Coleoptera (Othnonius batesi), and Diptera [Chironomus near decorus ( = attenuatus)], ans vertebrate proxvirus DNA (vaccinia, IHD-D) in cesium chloride gradients gave buoyant density values of 1.690 g/ml for vaccinia DNA and 1.676 to 1.681 g/ml for the entomopoxvirus DNAs. The molar fraction of guanine and cytosine (G+C) calculated from the buoyant density of entomopoxvirus DNA was 30 to 48% lower than the G+C values for vaccinia DNA. Thermal denaturation studies (Tm) of entomopoxvirus DNA confirmed the low G+C values and established that these DNAs were double-stranded in vitro. DNA base ratios calculated on the basis of buoyant density and Tm indicate that there are no abnormal bases present in the entomopoxvirus genome.