The intracellular distribution of p39, a 39,000-dalton substrate for a number of tyrosine protein kinases, has been determined by indirect immunofluorescence microscopy. No binding of anti-p39 antibodies to intact cells was observed, indicating that this protein is not accessible to antibody on the cell surface. Following detergent permeabilization of formaldehyde-fixed cells, a reasonably uniform cytoplasmic labeling was observed. This fluorescence was most pronounced in membrane ruffles, especially in the leading lamellae of migrating cells, and in areas of cell-cell contact. Brief permeabilization of cells with detergent prior to formaldehyde fixation resulted in the appearance of a reticular lattice. An identical staining pattern was observed when fluorescently-labeled lectins were used as plasma membrane markers, but not when antibodies to a variety of cytoskeletal proteins were used. Taken together, these results indicate that p39 is, at least in part, located at the cytoplasmic surface of the plasma membrane. Immunolabeling of Rous sarcoma virus-transformed cells with anti-p39 antibodies resulted in fluorescent staining patterns indistinguishable from those observed in untransformed cells. It is conceivable that p39 plays some structural role within a protein network underlying the plasma membrane.