Phosphotyrosine coupled to KLH, BSA, and OVA was used for the production and screening of antibodies to phosphotyrosine. 800 hybridomas secreting antibodies that bound to phosphotyrosine were detected by ELISA. The most reactive 100 of these 800 were tested subsequently for their ability to bind phosphotyrosine-containing proteins on Western blots. Eight stable hybridoma cell lines were selected for further study, cloned by limiting dilution, and grown as ascites. These antibodies were purified by three different methods, and it was found that affinity chromatography on phosphotyrosine-affigel provided the most rapid and effective method. Many phosphotyrosine-containing proteins were detected by using these antibodies in Western blotting and immunoaffinity purification procedures. Binding of anti-phosphotyrosine antibody could be competed by phosphotyrosine or phenylphosphate but not by phosphoserine, phosphothreonine, or free phosphate. These antibodies should be useful for the identification and purification of proteins phosphorylated on tyrosine residues in transformed and growth factor-treated cells.