We describe the use of graded decrements of medium osmolarity to progressively unmask respiratory capacities of whole human platelets in suspension. This departure led to the first demonstration that human platelet mitochondria are capable of tightly coupled respiration that responds to addition of mitochondrial substrates, ADP, and inhibitors in a way that other mammalian mitochondria are expected to behave. In 300 mosM media added alpha-glycerophosphate (G3P), succinate, or ADP effected only slight stimulation of base-line O2 consumption. At 180 mosM O2 consumption peaked and was not significantly affected by succinate or ADP. At 80 mosM base-line O2 consumption fell precipitously and was restored by G3P or succinate prior to being raised to its highest levels by ADP. Added NADH had no effect on O2 consumption at 80 mosM but sharply stimulated it when platelet suspensions were exposed to 60 mosM media by pretreatment with distilled water. At 80 mosM, selected compounds that inhibit or uncouple oxidative phosphorylation of isolated mammalian mitochondria from a variety of cells exerted similar influences on while platelets.