Vesicular stomatitis virus, grown on the WIL(2)-3A line of continuously growing human lymphocytes, contains DNA in addition to the viral RNA. By contrast, virus grown on BHK 21-C13 fibroblasts has no detectable DNA. The virus-associated DNA is found in both the B and T particles of the virus and is resistant to deoxyribonuclease. The DNA is intimately associated with the virus and appears to be incorporated into the viral ribonucleoprotein core structure produced by treatment of the virus with Nonidet P40 follow edby CsCl isopycnic banding. The virus-associated DNA has an isopycnic density of 1.699 g x cm(-) (-3) in CsCl, identical to that of human DNA. The average molecular weight of the DNA molecules associated with the virus is 9.0 x 10(5), as determined by velocity sedimentation in sucrose density gradients and studies of its contour length in the electron microscope. DNA.DNA reassociation kinetics of this DNA demonstrate that the DNA is of host origin and rules out the possibility that it originates from contaminating microorganisms or mitochondria. The analytical complexity of the virus-associated DNA shows that 50% of the virus-associated DNA sequences are homologous to the repeated DNA sequences of human DNA and the remaining 50% are homologous to human unique sequences. On the average, there is one molecule of DNA for each four virion particles.