Chromatin isolated from Ehrlich ascites tumor cells showed two DNA synthetic activities differing in sensitivity to N-ethylmaleimide. For studies on the nature of activities and relationship to kinetics of DNA polymerase, a new method was developed for detecting the activity of DNA polymerase released from chromatin DNA during DNA synthesis in vitro. The activity of DNA polymerase released was measured in a reaction mixture for DNA synthesis using exogenously added poly(dA-dT) as a template-primer in the presence of actinomycin D. Evidence that the DNA polymerase released was actually involved in DNA synthesis of chromatin was obtained in experiments using chromatin isolated from cells treated with various concentrations of 1-beta-D-arabinofuranosylcytosine and chromatin from adult mouse liver. The experiments showed that chromatin isolated from cells in which only small amount of DNA polymerase was engaged in DNA synthesis released a negligible amount of DNA polymerase, especially N-ethylmaleimide-sensitive polymerase. Kinetic analysis of DNA polymerase during chromatin DNA synthesis by the new method suggested that KCl at the optimal concentration (10-20 mM) for the N-ethylmaleimide-sensitive chromatin activity enhanced the binding of the N-ethylmaleimide-sensitive DNA polymerase to chromatin DNA. From the findings that addition of actinomycin D or omission of dNTPs from the preincubation mixture prevents this binding, it is suggested that the binding of DNA polymerase is followed by the DNA chain synthesis and that the DNA polymerase involved in this reaction is N-ethylmaleimide sensitive. Data on the effect of KCl on the rate of chromatin DNA synthesis and on the size of the DNA chain favor this assumption.