The phospholipid transmembrane distribution and movement in the plasma membrane of rainbow trout spermatozoa was determined with spin-labelled phospholipid analogues. After initial incorporation in the outer membrane leaflet, only the aminophospholipids, phosphatidylserine (PS) and phosphatidylethanolamine (PE) redistributed towards the inner leaflet. At equilibrium, more than 90% PS and approximately 80-85% PE were located in the cytoplasmic leaflet. The inward motion was significantly faster for PS (t1/2 approximately 5 min) than for PE (t1/2 approximately 60 min). Both the velocity and the extent of this redistribution were dependent on the cellular ATP level, arguing for the involvement of the aminophospholipid translocase. Comparison of the electron paramagnetic resonance spectra recorded from analogues located in either one of the leaflet showed an important difference of microviscosity between the cytoplasmic and the extracellular bilayer leaflets. Assuming that the analogue distribution reflects the asymmetry of the endogenous phospholipids, the higher probe mobility in the inner leaflet could be correlated to a higher degree of unsaturation of the fatty acids present in this hemi-leaflet.