Canine myocardial sarcolemma was purified, and its phospholipid constituents were determined by gas chro-matography-mass spectrometry, fast atom bombardment mass spectrometry, and conventional techniques. Canine myocardial sarcolemma contained 2.7 μmol of lipid Pi/mg of protein which was comprised predominantly of choline glycerophospholipids (47%), ethanolamine glycerophospholipids (28%), and sphingomyelin (11%). Sarcolemmal phospholipids contained 40% plasmalogen which was quantitatively accounted for by choline (57% of choline glycerophosphohpid) and ethanolamine (64% of ethanolamine glycerophosphohpid) plasmalogens. Choline plasmalogens contained predominantly the vinyl ether of palmitic aldehyde though ethanolamine plasmalogens were composed predominantly of the vinyl ethers of stearic and oleic aldehydes. The majority of sarcolemmal ethanolamine glycerophospholipids (75%) contained arachidonic acid esterified to the sn-2 carbon. Sphingomyelin was composed predominantly of long-chain saturated fatty acids (stearic and ara-chidic) as well as substantial amounts (8%) of odd chain length saturated fatty acids. The possible functional role of these unusual phospholipid constituents is discussed.