Ankyrin is a well characterized membrane skeletal protein which has been implicated in the anchorage of specific integral membrane proteins to the spectrin-based membrane skeleton in a number of systems. In this study, the organization of ankyrin was examined in lymphocytes in relation to T cell function. Light and electron microscope immunolocalization studies revealed extensive heterogeneity in the subcellular distribution of ankyrin in murine tissue-derived lymphocytes. While ankyrin can be localized at the lymphocyte plasma membrane, it can also be accumulated at some distance from the cell periphery, in small patches or in a single discrete, nonmembrane-bound structure. Double immunofluorescence studies demonstrated that ankyrin colocalizes with spectrin and with the signal transducing molecule protein kinase C beta (PKC beta) in tissue-derived lymphocytes, suggesting a functional association between these molecules in the lymphocyte cytoplasm. In addition, T lymphocyte activation-related signals and phorbol ester treatment, both of which lead to PKC activation, cause a rapid translocation of ankyrin, together with spectrin and PKC beta, to a single Triton X-100-insoluble aggregate in the cytoplasm. This finding suggests a mechanism for the reported appearance of PKC in the particulate fraction of cells after activation: activated lymphocyte PKC beta may interact with insoluble cytoskeletal elements like ankyrin and spectrin. Further evidence for a link between the subcellular organization of these proteins and PKC activity is provided by the observation that inhibitors of PKC activity cause their concomitant redistribution to the cell periphery. The dynamic nature of lymphocyte ankyrin and its ability to accumulate at sites distant from the plasma membrane are properties which may be unique to the lymphocyte form of the molecule. Its colocalization with PKC beta in the lymphocyte cytoplasm, together with its redistribution in response to physiological signals, suggests that structural protein(s) may play a role in signal transduction pathways in this cell type. Our data support the conclusion that ankyrin is not solely involved in anchorage of proteins at the plasma membrane in lymphoid cells.