Simian virus 40 (SV40) chromatin has a nucleosome structure similar to that of cellular chromatin and serves as a useful system for studying replication of cellular chromatin. A method for selectively permeabilizing African green monkey kidney cells (CV-1) with lysolecithin and DNA replication of SV40 chromatin in this permeable cell system are described. CV-1 cells treated with lysolecithin were markedly swollen but retained the basic morphology. This permeable cell system was successfully applied to the study of SV40 DNA synthesis as a single cell cycle event in CV-1 cells. SV40 DNA was synthesized with host polymerase α when supplied with appropriate substrates, which do not penetrate intact cells. Autoradiography of agarose gels indicated that replicating SV40 DNA completed replication in lysolecithin-treated permeable cells without the addition of cytosol. SV40 DNA of catenated dimers, both circules being nicked and gapped, were recovered from the agarose gel by freezing and squeezing gel slices.