In human KB cells permissively infected with adenovirus type 2, the high mol. wt. forms of virus DNA have been characterized. These size classes of virus DNA sediment at greater than 100S and 40 to 90S in alkaline sucrose density gradients. Considerable evidence from a series of earlier communications supports the notion that the high mol. wt. forms of virus DNA represent virus DNA sequences covalently linked to cellular DNA. 3H-labelled high mol. wt. adenovirus type 2 DNA from productively infected cells can be shown to hybridize to virus DNA fixed to filters. In the present paper we demonstrate that on alkali elution of the DNA from the filters used in the first step of the hybridization experiment, the labelled DNA re-hybridizes to cellular DNA in the second step of a sequential hybridization experiment. The order of performing the two successive hybridization experiments can be reversed and very similar results are obtained. These data provide conclusive evidence for the covalent linkage of virus and cellular DNA sequences in KB cells productively infected with adenovirus type 2.