Sulfhydryl and disulfide metabolism in platelet function has recently reemerged as a focus of platelet research. In this study we tested the effect of redox buffer on platelet aggregation and the effect of reduced glutathione (GSH) and platelet activation on sulfhydryl exposure in the platelet fibrinogen receptor, αIIbβ3. In the presence of subthreshold concentrations of agonist, physiologic concentrations of GSH (10 μM) stimulated platelet aggregation and secretion. These effects were found with more than one platelet agonist and with different low molecular weight thiols, including homocysteine. The effect of low molecular weight thiols was reproduced with the peptide LSARLAF which directly activates platelets through αIIbβ3, suggesting that the mechanism is at the level of this integrin. After determining optimal sulfhydryl labeling conditions for αIIbβ3 (5 mM EDTA, 37 °C, 60 min), we found that GSH (10 μM) generated sulfhydryls in the β3 subunit. To determine if the requirement was for reducing equivalents or for a redox potential (ratio of GSH to GSSG), aggregation was further studied with the addition of low concentrations of GSSG to the GSH. With a ratio of GSH/GSSG of 5/1, similar to that of blood, the addition of GSSG potentiated the stimulatory effect as compared to GSH alone. This indicates that, for potentiation of aggregation, GSH is not simply reducing disulfide bonds; there is rather a requirement for a certain redox potential. Additional studies performed in the absence of added glutathione showed an increase in sulfhydryl labeling in the β3 subunit during platelet activation. Finally, we show that vicinal dithiols of platelet surface proteins are involved in the sulfhydryl-dependent pathways of platelet activation. In summary, these data imply that the redox potential of blood regulates activation of the αIIbβ3 integrin and together with other reports in the literature suggest that disulfide bond cleavage with sulfhydryl generation in β3 is involved in activation of this receptor.