The adenovirus-specific DNA-binding protein was isolated from adenovirus type 5-infected KB cells and shown to possess DNase inhibitor activity. The protein decreased the rate of hydrolysis of single-strand DNA proportionately to its concentration in the reaction. Two peaks of activity were obtained upon sedimentation in a glycerol gradient, probably corresponding to the two major adenovirus-specific polypeptides in the preparation (molecular weights, 72,000 and 44,000). The DNase inhibitor activity of the adenovirus DNA-binding protein was distinguishable from that of the cellular DNA-binding protein, which we have described previously (K, Nass and G. D. Frenkel, J. Biol. Chem. 254:3407-3410, 1979), by its pattern of sedimentation and by the effect of temperature on the two activities. For the adenovirus DNA-binding protein, the ratio of DNase inhibitor activity at 43 degrees C to that at 30 degrees C was approximately 14, whereas for the cellular protein this ratio was less than 3. The DNase inhibitor activity with the temperature coefficient of 14 was absent from cells infected with adenovirus type 5 ts125 at 40 degrees C. DNase inhibition is a simple, sensitive, quantitative method for assay of the adenovirus DNA-binding protein.