DNA polymerase activities in uninfected KB cells or KB cells infected with adenovirus type 5 (Ad5) were compared by chromatography on DNA-cellulose and DEAE-cellulose and by isoelectric focusing. On DNA cellulose three components were found both in infected and in uninfected cells. The major component eluted at 0.15 M NaC1 and contained DNA polymerase alpha. Two minor components were found, one which did not bind to DNA-cellulose and one which bound strongly. This latter component contained DNA polymerase beta as characterized by DEAE-cellulose chromatography and sedimentation studies. No difference in properties between uninfected or Ad5-infected KB cells was found for the beta-polymerase. DEAE-cellulose chromatography of DNA polymerase alpha revealed the presence of two activities eluting at 0.11 and 0.13 M NaC1 designates as alphaI and alphaII, respectively. In Ad5-infected cells alphaII was the major component. In uninfected, stationary cells alphaI was the major component and alphaII was only detectable as a shoulder in the elution profile. However, fast growing, uninfected cells gave a similar pattern as Ad5-infected cells. These results indicate that the observed change of the DNA polymerase pattern after infection with Ad5 is related to the level of DNA synthesis and not to the induction of a viral enzyme.