Cyclosporin A (CsA) was incubated at concentrations of 5.0 x 10⁻⁶ M for 72 hours, and at concentrations of 1.0 and 0.5 x 10⁻⁶ M for 30 days with kangaroo rat proximal tubular epithelial cells (PtK₂) in order to evaluate its effects on the cytoskeleton. Alterations in the cytoskeleton were assessed by indirect immunofluorescence of viable cells, and by two dimensional electrophoresis of a high salt extract from the cells. There is a selective alteration of the cytokeratin intermediate filament organization in both the short term (5 x 10⁻⁶ M, 72 hr) and long term (1 and 0.5 x 10⁻⁶ M, 30 days) exposures. There are either peri-nuclear rings formed or the formation of a single aggregate clump of the cytokeratins within the cytoplasm. Other components of the cytoskeleton, the microtubules and the microfilaments remain unaffected at both short term and long term exposures. Along with this cytokeratin alteration in CsA exposed cells is the decrease or elimination of an acidic triplet of cytokeratin protein monomers, human equivalent K15 (50 kd), K16 (48 kd), K17 (46 kd). This may be related to CsA-associated nephrotoxicity.