The effect of aggregating agents on the hexose monophosphate shunt (HMPS) and the relationship of 14CO2 production to platelet aggregation were studied in normal volunteers. Platelets collected in ACD were suspended in modified Ringer’s bicarbonate buffer without washing and were studied before and after the addition of collagen, adenosine diphosphate (ADP), epinephrine, or thrombin. HMPS and Kreks cycle activities were estimated by the yields of 14CO2 from glucose-1-14C (C1) and glucose-6-14C (C6). 14CO2 production from each substrate was measured continuously during experiments using paired, vibrating reed electrometers and incubation flasks. Both flasks contained aliquots of the same platelet suspension. Baseline 14CO2 production averaged 22 ± 4.5 mµmoles/hr/109 platelets from C6 as compared to 33 ± 6 mµmoles/hr/ 109 platelets from C1. Each aggregating agent gave a prompt and striking increase in 14CO2 production from C1. In contrast, the increase in 14CO2 production from C6 was not detectable for 10 min and then production increased slowly. Inhibition of 14CO2 nate (25 mM) did not interfere with platelet aggregation. Stimulation of 14CO2 production from C1 by aggregating agents was unaffected by malonate. These data indicate that platelet aggregation coincides with stimulation of the HMPS, but the increase in Krebs cycle activity occurs later and is not essential for platelet aggregation.