The fatty acid composition of individual phospholipids in subcellular fractions of sheep platelets and the asymmetrical distribution of phosphatidylethanolamine (PE) fatty acyl chains across the plasma membrane were examined. The main fatty acids of total lipid extracts were oleic (18:1; 32-41%), linoleic (18:2, 10-17%), stearic (18:0; 13-15%), palmitic (16:0; 11-15%) and arachidonic (20:4; 8-12%) acids, with a saturated/unsaturated ratio of about 0.4. Each phospholipid class had a distinct fatty acid pattern. Sphingomyelin (SM) showed the highest degree of saturation (50%), with large proportions of behenic (22:0), 18:0 and 16:0 acids. The main fatty acid in PE, phosphatidylserine (PS) and phosphatidylcholine (PC) was 18:1n-9. Our findings suggest that fatty acids are asymmetrically distributed between the choline versus the non-choline phospholipids, and also between plasma membranes and intracellular membranes. The transbilayer distribution of PE fatty acids in plasma membranes from non-stimulated sheep platelets was investigated using trinitrobenzene-sulfonic acid (TNBS). A significant degree of asymmetry was found, which is a new observation in a non-polar cell. The PE molecules from the inner monolayer contained higher amounts of 18:2 and significantly less 18:1 and 20:5 than those found in the outer monolayer, although no major differences were detected in the transbilayer distribution of total unsaturated versus saturated PE acyl chains.