T84 cells, a human intestinal epithelial cell line, serve as a model of electrogenic Cl- secretion. We find that cAMP-elicited Cl- secretion in T84 cells is accompanied by a marked redistribution of F-actin in the basolateral portion of the cell. To prevent this F-actin redistribution and thereby assess its importance to Cl- secretion, we have defined simple conditions under which this model epithelium can be loaded with nitrobenzoxadiazole (NBD)-phallicidin. This reagent binds F-actin with high affinity thus stabilizing the F-actin cytoskeleton by preventing depolymerization, an event necessary for dynamic reordering of actin microfilaments. NBD-phallicidin loading is not cytotoxic as assessed by lactic dehydrogenase release, protein synthesis, transepithelial resistance, and the ability of the loaded cells to pump Na+ in an absorptive direction in response to the apical addition of a Na+ ionophore. However, cAMP-elicited redistribution of F-actin and the cAMP-elicited Cl- secretory response are both markedly impaired in NBD-phallicidin preloaded T84 cells. In contrast, the carbachol-elicited Cl- secretory response (Ca++ mediated) is not attenuated by NBD-phallicidin preloading nor is it accompanied by redistribution of F-actin. These findings suggest that the cAMP-elicited cytoskeletal redistribution we describe is an integral part of cAMP-elicited Cl- secretion in T84 cells.