The subcellular localization of the 34 kd protein substrate of the pp60src kinase was investigated by immunofluorescence microscopy and subcellular fractionation. When permeabilized fibroblasts were stained with a monoclonal anti-34 kd protein antibody, a diffuse reticular pattern was observed. The 34 kd protein was not exposed on the outside surface of the cell. Double immunofluorescence staining experiments established that the 34 kd protein distribution was similar to that of the membrane-associated protein alpha-spectrin. The 34 kd protein was found in cell sections to be concentrated along the cell edges. Taken together, these results suggested that the 34 kd pp60src substrate was associated with the inside surface of the plasma membrane. This conclusion was supported by subcellular fractionation experiments in which the 34 kd protein was observed to fractionate with the plasma membrane. These localization studies support further the hypothesis that many of the primary effects of the pp60src kinase occur at the plasma membrane.