Simian virus 40 (SV40) infection of monkey kidney cells induces successive rounds of cellular DNA synthesis without intervening mitosis. To gain an understanding of the mechanisms responsible for disruption of cell cycle control during lytic infection, pRB phosphorylation and cell cycle distribution were examined following SV40 infection of CV-1 cells. The hypophosphorylated pRB present in confluent CV-1 cells was phosphorylated within 14 h following SV40 infection. Phosphorylated pRB then remained the predominant form as cells progressed from late G1 through S phase. Hypophosphorylated pRB reappeared as cells moved through G2 and acquired a tetraploid (> G2) DNA content. The reappearance of hypophosphorylated pRB in a population with decreasing numbers of cells in G1 phase and increasing numbers of cells in > G2 suggests that accumulation of hypophosphorylated pRB may be involved in T antigen-induced tetraploidy.