A gene encoding the major inner capsid protein, VP6, of avian rotavirus was inserted into the eukaryotic expression vector pAX-91 under the control of the SR alpha promoter and was expressed at a high level in simian COS7 cells. The expressed VP6 was indistinguishable in terms of electrophoretic mobility from the corresponding protein synthesized in simian MA104 cells infected with avian rotavirus. Binding assays with a series of monoclonal antibodies (mAbs) that corresponded to four antigenic sites on VP6 of avian rotavirus showed that the antigenic characteristics of the expressed product were identical to those of the native VP6 of avian rotavirus virions. Fiber-like structures that reacted strongly with antiserum against rotavirus were observed in VP6-expressing COS7 cells. Furthermore, an analysis of the tertiary structure of the expressed VP6 protein indicated that it adopts a trimeric configuration, similar to that of the major inner capsid protein of PO-13 virus. From these results, it appears that recombinant VP6 will facilitate studies of the structure and function of authentic VP6, an important protein in avian rotavirus.