The best characterized of the autonomous parvoviruses are those of rodent origin. The purpose of this study was to examine the physicochemical characteristics of the genome of an autonomous parvovirus of non-rodent origin, bovine parvovirus (BPV). BPV was isolated from infected cells by centrifugation through a sucrose-CsCl step-gradient. The virion DNA was released from the capsids by alkali treatment. Upon chromatography on hydroxyapatite, two peaks of radioactivity were consistently observed. Peak I eluted at a sodium phosphate concentration of 0.17 M, intermediate between single- and double-stranded DNA markers. Peak II eluted in the same position as double-stranded DNA. The DNA eluting in peak I represented the viral genome. About 7% of the genome length was found to exist as a duplex. The BPV DNA was found to have sedimentation coefficients of 16.5 Sand 27 S in alkaline and neutral sucrose, respectively. These S values corresponded to a molecular weight of approximately 1.7 x 10⁶ daltons. The BPV DNA was rich in thymine (31.3%) and had a GC content of 45.1%. The buoyant density of the single-stranded BPV genome was determined to be 1.721 g/cm³. The DNA eluting in peak II was shown by hybridization to be of BPV origin. Furthermore, the peak II DNA was shown to be of the same size as the single-stranded BPV genome, by sedimentation in alkaline sucrose. Electron microscopy revealed that the peak II DNA was linear and double-stranded. It is concluded that BPV packages the plus strand into separate virions in significant amounts, resulting in the reannealing of complimentary plus and minus strands upon release from the virions during isolation of the DNA / M.S.