The DNA binding proteins in a nuclear membrane fraction that can synthesize DNA in vitro (referred to as "nuclear membrane complex") and in the cytoplasm of adenovirus infected and uninfected cells were isolated and characterized. Suspension cultures of human KB cells infected with human adenovirus 2 were treated with 25 mu-g/ml of arabinosylcytosine starting at 2 hr to block the synthesis of viral structural proteins, and then labeled with (3H)leucine from 6 to 24 hr after infection. Uninfected cells were treated similarly and labeled with (14C)leucine. The 3H-labeled proteins (infected cells) and 14C-labeled proteins (uninfected cells) isolated from the cytoplasm were mixed, as were the corresponding proteins isolated from the membrane complex, and each mixture was fractionated by stepwise elution from single-stranded DNA-cellulose columns. From 50 to 60% of the labeled protein in the membrane complex from infected cells and 40 to 50% of that from uninfected cells bound to DNA-cellulose in 0.05 M NaCl. Much less protein from the cytoplasm was bound to DNA cellulose, 20% from infected cells and 11% from uninfected cells. Gel electrophoresis of the mixture of 3H- and 14C-labeled proteins eluted from DNA-cellulose by different concentrations of NaCl revealed the following. (1) The 0.15 and 0.40 M NaCl eluates from the membrane complex of infected and uninfected cells contained a heterogenous mixture of similar polypeptides. (2) The 0.6 M NaCl eluate from the membrane complex derived from infected cells contained two major DNA binding proteins with molecular weights of 75,000 and 45,000 that were absent from uninfected cells. Large quantities of these two proteins were present in highly purified form in the 0.6 M NaCl eluate from the cytoplasm of infected cells. The DNA binding proteins of molecular weight 75,000 and 45,000 that are present in the cytoplasm are identical with those present in the membrane complex, as established by coelectrophoresis. (3) Two major cell-specific proteins of molecular weight 40,000 and 15,000-17,000 were present in the 2 M NaCl eluate of the membrane complex from uninfected and infected cells. A major cell-specific protein of molecular weight 33,000 was present in the 0.15 and 0.4 M NaCl eluates of the uninfected and infected cell cytoplasmic fractions. Aanalysis of cells labeled at 2-6 hr after infection in the absence of arabinosyl cytosine indicated that the synthesis of the DNA binding proteins of molecular weight 75,000 and 45,000 begins early after infection prior to the onset of viral DNA replication.