After infection of RNA tumor viruses to susceptible cells, viral RNA is reversely transcribed into proviral DNA. In order to disclose the site of proviral DNA synthesis in the cells, chick embryo fibroblasts (CEF) were enucleated by centrifugation in the presence of cytochalasin B, and the enucleated CEF (cytoplasts) were infected with B77 strain of avian sarcoma virus (B77-ASV). Incorporation of (3)H-thymidine into DNA in the cytoplasts was investigated by autoradiography. Photopositive grains were observed in cytoplasts infected with B77-ASV, but not in mock-infected cytoplasts. The photopositive grains in the cytoplasts infected with B77-ASV disappeared almost completely by DNase I treatment. N-demethyl rifampicin, which is a specific inhibitor of reverse transcriptase, inhibited the appearance of photopositive grains. The B77-ASV-infected cytoplasts were ultrathinsectioned for electron microscopic autoradiography. The photopositive grains appeared in the cytoplasm without relation to mitochondria. These results indicate that the proviral DNA synthesis is initiated in the cytoplasm of B77-ASV- infected chick embryo fibroblast without the direct participation of nucleus.