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Kilham rat virus (KRV) was found to grow in a rat nephroma cell line and to form plaques on secondary rat embryo monolayers. The virus was purified by enzymatic treatment and isopycnic cesium chloride sedimentation. KRV bands at a density of 1.41 g/cm3 in cesium chloride. It contains about 26.5% deoxyribonucleic acid (DNA). The sedimentation coefficient S2o, in sucrose gradients was 122 corresponding to a molecular weight of 6.6 X 106 daltons. The reaction of formaldehyde with the KRV virion suggests that the DNA in situ is single-stranded. DNA extracted from KRV had a buoyant density of 1.715 g/cm' in cesium chloride. The S20,W was determined in sucrose gradients to be 16, and the molecular weight was calculated to be approximately 1.7 x 106 daltons. The base composition of the DNA is 26.7% adenine, 30.8% thymine, 20.0% guanine, and 22.5% cytosine. On the basis of its noncomplementary nucleotide ratio, melting curve, and the reaction with formaldehyde, the DNA of KRV is believed to be single-stranded.