p81, a protein-tyrosine kinase substrate previously identified in epidermal growth factor-treated A431 cells, is demonstrated to be homologous to ezrin, an 80-kD component of microvillar core proteins. p81 has been characterized using antiserum raised against purified chicken intestinal ezrin. p81, located by indirect immunofluorescent staining, is concentrated in surface projections of A431 cells such as microvilli and retraction fibers. None of the conditions of biochemical cell fractionation tested completely solubilizes p81; the insoluble p81 partitions as if associated with the cytoskeleton. The soluble form of p81 behaves as a monomer in all extraction procedures studied. EGF-stimulated phosphorylation of p81 does not appear to change its intracellular location. p81 exhibits a wide tissue distribution with highest levels of expression in small intestine, kidney, thymus, and lung. Intermediate levels are found in spleen, thymus, lymph nodes, and bone marrow, with low levels in brain, heart, and testes. p81 is undetectable in muscle and liver. In A431 cells, p81 is phosphorylated on serine and threonine residues. Upon EGF treatment, approximately 10% of p81 becomes phosphorylated on tyrosine, and the phosphorylation of threonine residues increases.