It has been suggested that avian reovirus sigma 3 protein is analogous to sigma 1 trimer, the mammalian reovirus attachment protein. We have investigated the multimeric nature and cell binding ability of sigma 3 protein. The data presented here demonstrate that sigma 3 protein is a multimer in its undisrupted form as determined by SDS-PAGE in non-dissociating conditions. However, virion-associated sigma 3 protein and COS-7 cell-expressed protein behaved differently in SDS-PAGE, suggesting a need for virus-associated factor(s) to control the multimerization of the protein. The data also show that Escherichia coli expressed sigma 3 fusion protein (sigma 3-MBP) in its multimeric form is capable of attaching to Vero cells. The binding was found to be specific and receptor mediated by the fact that it was inhibited by a monoclonal antibody specific for sigma 3 protein and by competition with avian reovirus particles. As determined by a reverse experiment, sigma 3-MBP was also able to reduce the virus p.f.u. in monolayer cell cultures, indicating the important role of sigma 3 protein in the initiation of virus infection.