Spin-labeled analogues of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin were added to human platelet suspensions. Due to the partial water solubility of these spin-labeled lipids which possess a relatively short beta-chain (C5), they incorporate rapidly in membranes. The orientation of the spin-labels within the platelet plasma membrane was assessed by following the spontaneous reduction at 37 and 4 degrees C due to endogenous reducing agents present in the cytosol. The rate of spontaneous reduction showed unambiguously that the labels incorporated initially in the outer leaflet of the plasma membrane and that the rate of outside-inside translocation of the aminophospholipids was faster than that of the choline derivatives. For example, at 37 degrees C, the half-time for the transverse diffusion of a phosphatidylcholine analogue was found to be of the order of 40 min, while it was less than 7 min for the phosphatidylserine analogue. At low temperatures, a fraction of the labels gave rise to a strongly immobilized ESR component. This fraction, which corresponded to 20-30% of the initial spin-label concentration, was found resistant to chemical reduction from the inner side of the membrane and also to externally added reducing agents such as ascorbate. Presumably these immobilized lipids are trapped in a gel phase formed in the outer leaflet at 4 degrees C. Cell aging, which depletes the cells of ATP, resulted in the progressive inhibition of the fast transport of the aminophospholipids from the outer to inner leaflet. Treatment of the cells with iodoacetamide completely blocked the transverse diffusion of the spin-labels.(ABSTRACT TRUNCATED AT 250 WORDS)