We have developed a rapid in situ screening procedure that enables prescreening of hundreds of hybridomas in a 96-well format. The procedure involves fluorescence immunostaining of cells cultured in 96-well plates and the use of a fluorescence plate reader to detect reactive antibodies. Positive immunostaining in individual well, as denoted by elevated readings, is then confirmed by fluorescence microscopy. Using the method described here, we have successfully identified monoclonal antibodies that are specific to the nuclear receptor, peroxisome proliferator-activated receptor gamma (PPAR gamma). This assay is readily applicable for screening hybridomas raised against cell surface or intracellular antigens to aid in the initial identification of antibodies reactive in immunocytochemical procedures.