Pretreatment of human platelets with the metabolic inhibitors rotenone and 2-deoxyglucose, before French press homogenization, has led to the isolation of dense storage granules in an overall yield of about 20%. The concentrations of serotonin, ATP and ADP were estimated in the dense granules. Serotonin was 40--60-fold enriched in the dense granules compared to the platelet homogenate. Stored ATP and ADP were also 40-fold enriched in the dense granules compared to the estimated storage nucleotide pool in intact platelets. The ATP to ADP ratio in the isolated dense granules was 0.68-0.70, the same as the ratio of the secreted ATP and ADP. In platelets prelabeled with [3H]adenine, the specific radioactivities of the ATP and ADP in the isolated dense granules and of the secreted ATP and ADP were both negligible, whereas the estimated specific radioactivity of the metabolically active ATP and ADP was 2,000 cpm/nmol. These results confirm that the ATP and ADP in the isolated dense granules are the same as the secreted ATP and ADP in terms of metabolic inactivity and their ATP to ADP ratios.