Platelet surface thiols and disulphides play an important role in platelet responses. Agents that reduce disulphide bonds expose the fibrinogen receptor in platelets and activate the purified glycoprotein (GP) IIbIIIa receptor. Protein disulphide isomerase (PDI), an enzyme that rearranges disulphides bonds, is found on the platelet surface where it is catalytically active. We investigated the role of PDI in platelet responses using (1) rabbit anti‐PDI IgG specific for PDI, (2) a competing substrate (scrambled ribonuclease A), and (3) the PDI inhibitor, bacitracin. Fab fragments of the rabbit anti‐PDI IgG inhibited platelet responses to the agonists tested (ADP and collagen), whereas Fab fragments prepared identically from normal rabbit IgG had no inhibitory effect. Scrambled ribonuclease A blocked platelet aggregation and secretion, but native ribonuclease A did not. When biphasic platelet aggregation was examined using platelets in citrated plasma, the principle effect of bacitracin was on second phase or irreversible aggregation responses and the accompanying secretion. Using flow cytometry and an antibody specific for activated GPIIbIIIa (PAC‐1), the rabbit anti‐PDI Fab fragments substantially inhibited activation of GPIIbIIIa when added before, but not after, platelet activation. In summary, we have demonstrated that protein disulphide isomerase mediates platelet aggregation and secretion, and that it activates GPIIbIIIa, suggesting this receptor as the target of the enzyme.