Mutation of the retinoblastoma (Rb) gene is found frequently in human sarcomas, lung, bladder and breast carcinomas and is the molecular basis for hereditary predisposition to retinoblastoma. The Rb protein is a nuclear phosphoprotein that is differentially phosphorylated during the cell cycle. Its precise function is unknown but it has been suggested that it may act as a transcriptional regulator or as a regulator of cellular DNA synthesis. The Rb protein forms specific complexes with the oncogenes of three different groups of DNA tumour viruses. We have prepared a new monoclonal antibody to the Rb protein and used it to establish sensitive immunoassays for Rb complexed to T antigen. In SV40-infected and transformed cells these assays showed that Rb enters a trimolecular complex containing p53, Rb and T. A large panel of human tumour cell lines was tested for expression, cellular localization and T-binding activity of Rb using the new antibody.