Free sulfhydryl and disulfide groups of an acid-soluble and acid-precipitable fraction and of isolated plasma membranes were quantified in fresh and stored human platelets. Fresh platelets contained 26.6 x 10-8 moles of total thiols including free sulfhydryl and disulfide groups per 109 cells. One-third of these thiols were present as disulfides. Acid-soluble thiols accounted for one-tenth of the total platelet thiols. Storage of platelets produced a reduction in the level of acid-soluble sulfhydryl and disulfide groups which was rapid during the first 24 hr and was precipitous in platelets stored at 22°C. Free sulfhydryl groups of the acid-precipitable fraction and of isolated platelet membranes showed a relatively rapid decrease during the first 24 hr of storage and declined thereafter very slowly. The disulfide levels of these cell fractions showed a reciprocal increase which complemented the fall in the number of free sulfhydryl groups. The storage temperature did not significantly influence these changes in protein thiols and disulfides. The coincidence of loss of platelet functions and of severe decrease in the level of free sulfhydryl groups during platelet storage at room temperature may suggest a cause-effect relationship between the two.