Acyl chain perdeuterated dimyristoylphosphatidylcholine (DMPC-d54) and dimyristoylphosphatidylserine (DMPS-d54) were incorporated by incubation into human erythrocytes. Light microscopic analysis demonstrated that erythrocytes incubated with DMPC-d54 became echinocytic while those incubated with DMPS-d54 became stomatocytic. This indicates that DMPC-d54 was incorporated preferentially into the outer monolayer whereas DMPS-d54 was selectively incorporated into the inner monolayer. Fourier transform infrared (FTIR) spectroscopy was used to monitor the conformational order of the incorporated phospholipids. The asymmetric CD2 stretching frequency of the inserted perdeuterated acyl chains was measured in both isolated membranes and intact erythrocytes as a function of temperature. DMPC-d54 incorporated into erythrocytes exhibited a cooperative phase transition at approximately 19 degrees C, i.e., at the same temperature as pure vesicles. In contrast, DMPS-d54 incorporated into red cells exhibited no phase transition, but possessed conformational order similar to that of the liquid-crystalline state. These results suggest that DMPC-d54 persists in domains in the outer monolayer while DMPS-d54 is dispersed in the inner monolayer. These experiments are the first to demonstrate that FTIR spectroscopy can be utilized to monitor directly a specific species of lipid molecule from the entire phospholipid population.