Amphiphilic moieties such as lysophosphoglycerides and long-chain acyl carnitines accumulate in ischemic myocardium and potentially contribute to the sequelae of myocardial ischemia. To characterize alterations in membrane molecular dynamics produced by amphiphilic compounds, highly purified preparations of canine myocardial sarcolemma were spin-labeled with paramagnetic probes (5-, 12-, or 16-doxyl stearate), and alterations produced by amphiphilic compounds were quantified by electron spin resonance spectroscopy. Incorporation of 1.5, 3, or 6 mol % palmitoyl lysophosphatidylcholine resulted in a decrease of the order parameter of 16-doxyl stearate from 0.164 to 0.161, 0.155, and 0.145, respectively. Similar increases in membrane fluidity in the interior of the bilayer were present when palmitoyl lysophosphatidylethanolamine, L-palmitoyl carnitine, and platelet-activating factor were incorporated into sarcolemma. In contrast, incubation of sarcolemma with lysophosphatidylcholine did not result in significant change of the order parameter of 5-doxyl stearate, even at 6 mol %, demonstrating that lysophosphatidylcholine increases the transmembrane fluidity gradient. Sarcolemma treated with phospholipase A2 exhibited a time-dependent decrease in the rotational correlation time and order parameter when lysophospholipids constituted a small amount (6%) of sarcolemmal phospholipids. Furthermore, the effects of lysophosphatidylcholine were not dependent upon its physical state, since bilayers composed of gramicidin and lysophosphatidylcholine resulted in similar increases in membrane fluidity as micellar lysophosphatidylcholine. The results suggest that alterations in sarcolemmal molecular dynamics are one mechanism through which amphiphilic moieties mediate their multiple effects. Such alterations could contribute to the electrophysiological and biochemical sequelae of myocardial ischemia.