Serotype-specific differences in the capacity of reovirus strains to inhibit proliferation of murine L929 cells correlate with the capacity to induce apoptosis. The prototype serotype 3 reovirus strains Abney (T3A) and Dearing (T3D) inhibit cellular proliferation and induce apoptosis to a greater extent than the prototype serotype 1 reovirus strain Lang (T1L). We now show that reovirus-induced inhibition of cellular proliferation results from a G(2)/M cell cycle arrest. Using T1L x T3D reassortant viruses, we found that strain-specific differences in the capacity to induce G(2)/M arrest, like the differences in the capacity to induce apoptosis, are determined by the viral S1 gene. The S1 gene is bicistronic, encoding the viral attachment protein sigma1 and the nonstructural protein sigma1s. A sigma1s-deficient reovirus strain, T3C84-MA, fails to induce G(2)/M arrest, yet retains the capacity to induce apoptosis, indicating that sigma1s is required for reovirus-induced G(2)/M arrest. Expression of sigma1s in C127 cells increases the percentage of cells in the G(2)/M phase of the cell cycle, supporting a role for this protein in reovirus-induced G(2)/M arrest. Inhibition of reovirus-induced apoptosis failed to prevent virus-induced G(2)/M arrest, indicating that G(2)/M arrest is not the result of apoptosis related DNA damage and suggests that these two processes occur through distinct pathways.