Immunodecoration of proteins containing phosphotyrosine residues (Tyr-P) was carried out by incubating electroblots of SDS gels with sheep antibodies that bind Tyr-P. Peptide-antibody complexes are detected with affinity-purified anti-sheep IgG conjugated to horse- radish peroxidase. When the procedure was tested with proteins having only phosphorylated serine or threonine sites, no appreciable reaction was observed, indicating the specificity of the method for Tyr-P sites This immunodetection procedure identified several autophosphorylated growth factor receptor tyrosine kinases (epidermal growth factor (egf) and insulin receptors) on electroblots of tissue extracts as well as phosphotyrosyl-proteins that were modified after cells were exposed to interleukin (IL)-3. In addition, immunoadsorption of Tyr-P- containing proteins was accomplished using anti-phosphotyrosine antibodies immobilized to Protein A-Sepharose or linked covalently to CNBr-activated Sepharose. This approach allowed semi- quantitative adsorption of phosphorylated egf-and insulin- receptors solubilized tissue preparations. Studies have been initiated to prepare phosphopeptides comprising phosphorylation sites of several important cellular proteins (e.g. receptors and cytoskeletal proteins) thought to be phosphorylated during cell activation. Novel procedures have been developed for the chemical phosphorylation of serine and threonine residues in these peptides. Specific antibodies against these sites may prove useful in characterizing the physiologic response of cells, especially in lymphocytes and neural tissue, to immuno- or neuro- modulatory influences.