Antiphosphotyrosine blotting is a technique for detecting tyrosine-phosphorylated substrates by the use of antibodies that recognize these residues on a wide variety of proteins. This unit describes conditions for cell lysis and immunoprecipitation of proteins with an antiphosphotyrosine antibody, followed by electrophoretic separation, immunoblotting, and color detection of the blotted proteins. This combination of steps provides particularly sensitive conditions for the detection of tyrosine-phosphorylated substrates, but also gives good results for any protein transferred to nitrocellulose, including whole-cell lysates or proteins immunoprecipitated with another antibody. Although the alkaline phosphatase color-detection system has the advantage of providing superior resolution and higher sensitivity without the use of any radioactivity, the (125)I-labeled Staphylococcus protein A detection system is described for use in conjunction with the blotting protocol.