A novel method has been developed for the initial screening of hybridomas produced against cell surface antigens. Glutaraldehyde-fixed cells were immobilized as targets on the lid of a 96-well tissue culture plate which had been precoated with poly-l-lysine. Antibody binding was determined by an immunoenzymatic method in an arrangement permitting both macro- and microscopic examination. After optimization with mouse thymus cells using existing rat monoclonal antibodies, new rat-mouse hybridoma cell lines against mouse thymocytes and bone marrow cells were screened. The antibodies could be characterized immediately both by the localization of the immune reaction (surface or intracellular) or as estimated by the frequency of positive cells recognized by the antibody in the sample. © 1992.