The formation of double-stranded viral DNA was examined in synchronized cells infected with minute virus of mice in early G1 phase. In the infected cells, a minimum of 50-100 copies of the input single-stranded DNA have been converted to a double-stranded form by mid S phase. In well-synchronized cells, the amount of double-stranded form by mid A phase. In well-synchronized cells, the amount of double-stranded viral DNA detected during G1 is on the order of a few copies per cell or less. When cells are infected in the presence of the thymidine analog, 5-bromodeoxyuridine, viral DNA synthesis is inhibited. However, 5-bromodeoxyuridine does not inhibit host DNA synthesis nor does it prevent replication of viral DNA if added to the infected cells in late S phase. Viral DNA replication first becomes resistant to 5-bromodeoxyuridine inhibition at the beginning of S phase. As 5-bromodeoxyuridine appears to specifically block early steps in viral DNA synthesis but not the subsequent replication of the DNA, the conversion of the input viral genome to a double-stranded form which undergoes further replication appears to be a S phase-specific event.