Hybrid hybridomas (tetradomas) producing bispecific monoclonal antibodies reacting with both horseradish peroxidase (HRP) and human alpha-fetoprotein (AFP) were obtained by fusing two hybridoma lines and selecting the fused cells by a fluorescence activated cell sorter (FACS III). The hybridoma cells were labelled before fusion with fluorescein isothiocyanate (FITC) and tetramethyl rhodamine isothiocyanate (TRITC) respectively and heterofluorescent cells were sorted out after fusion. Several clones were found to produce bispecific antibodies, and one clone, designated T1, was subjected to growth in ascitic fluid in mice in order to obtain large quantities of hybrid antibodies. Bispecific antibodies could be separated from the monospecific antibody populations by one-step hydroxylapatite chromatography. SDS-polyacrylamide gel electrophoresis demonstrated that the hybrid antibody molecules contained the heavy chains of both anti-HRP and anti-AFP origin. The bispecific antibodies were used to build up a sensitive two-site binding enzyme immunoassay.