The ability of HSV-1 DNA to become associated with host cell DNA in an alkaline-stable form has been demonstrated following infection of a ts baby hamster kidney growth mutant (ts BTN-1), at the non-permissive temperature (39.5 depgrees C). After 8 h pre-incubation at 39.5 degrees C, ts BTN-1 cells infected at this temperature using m.o.i. ranging from 0.5 to 200 p.f.u./cell fail to replicate virus DNA even though transport of input virus genomes to the nucleus is the same at both permissive and non-permissive temperatures. Virions containing 3H-labelled DNA were used to infect ts BTN-1 cells at 39.5 degrees C, and the total cellular DNA isolated from these cells was resolved into host and virus material by repeated CsCl equilibrium gradient centrifugation. A significant amount of the input radioactivity was found as a distinct band in the host region in both neutral and alkaline CsCl gradients, strongly suggesting a covalent association between host and virus DNAs. Evidence for this association was strengthened by demonstrating that radioactive material (virus DNA) banding in the host region of CsCl gradients could be driven towards the density expected for virus DNA following degradation of the putative hybrid molecules by shearing.