Agents that act as anion channel blockers (ACBs) and do not permeate cells appear to inhibit exocytosis in platelets, parathyroid cells, and neutrophils. Based in large part on these observations, anion influx through plasma membrane channels has been considered a factor controlling cellular secretion, but there have been no direct anion influx measurements in cells or granules to support this concept. We have found that ACBs inhibit only thrombin-induced platelet secretion, not secretion induced by ADP, collagen, or A23187. ACBs inhibit thrombin esterolytic activity, binding of thrombin to platelets, and thrombin-stimulated platelet production of malondialdehyde in proportion to the degree of inhibition of thrombin-induced platelet secretion. Thus inhibition of platelet secretion by ACBs is due to inactivation of the stimulatory agonist, thrombin, and not to interference with cellular secretion per se. We have also found that previously reported inhibition of secretion of parathyroid cells and neutrophils by ACBs can be explained by the ability of ACBs to interfere with detection of the cellular secretory products that were measured to assess exocytosis. Our measurements of parathyroid hormone and beta-glucuronidase in the presence of ACBs were reduced to the same degree as the reported reduction in apparent cellular secretion produced by these agents. We conclude that plasma membrane anion channels of the type that can be blocked by ACBs such as 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid, suramin, and probenecid do not participate in cellular secretory processes. Whether other types of anion channels exist that are not affected by these ACBs and whether there are mechanisms of anion flux during secretion not dependent on channels remain open questions.