Cell cultures derived from trabecular meshworks of human and bovine eyes and from bovine vascular endothelia were incubated at 37 degrees C for 1 hr with ethacrynic acid (ECA, 0.1-0.5 mmol/l) dissolved in culture medium. At 2 hr after the initial exposure, ECA at concentrations up to 0.4 mmol/l induced a reversible alteration in cell shape in all three cell types that was coincident with a change in the staining pattern of major cytoskeletal components including actin, alpha-actinin, vinculin, and vimentin. Distinct progressive alterations in beta-tubulin also occurred, with initial changes observed 10 min after ECA exposure. The ECA-induced changes in tubulin were blocked in part by preincubation with taxol (which stabilizes the microtubule structure), but they appeared to differ from those occurring with nocodazole (which interferes with tubulin assembly). These results suggest the possibility that ECA-induced increases in outflow facility may be mediated by alterations in the cytoskeletons of outflow pathway cells.