Anthryl-labeled fluorescent probes closely mimicking phosphatidylcholine and sphingomyelin were applied to study the state of these phospholipids in the rabbit erythrocyte membrane. At normal cholesterol levels both probes exhibited higher fluorescence polarization values in the membranes than in phospholipid vesicles of similar lipid composition, indicating a decreased fluidity of the probe environment in erythrocyte ghosts. In ghosts prepared from normal erythrocytes no evidence of lateral separation of phosphatidylcholine and sphingomyelin was found. At higher cholesterol levels, however, these lipids appear to segregate. Probably the effect of cholesterol on the erythrocyte membrane lipids involves lipid-protein interactions. At physiological concentrations, prostaglandin E1only weakly affects the state of phosphatidylcholine and sphingomyelin in erythrocyte membranes. Cholesterol enrichment amplifies the effect of prostaglandin E1. Although the prostaglandin E1-induced changes depended much upon whether the ghosts were enriched with cholesterol in vitro or in vivo, with both types of ghosts effects of prostaglandin E1were seen at extremely low effector concentrations that may have presented a few molecules of prostaglandin per ghost. The structural and functional significance of these findings is discussed. © 1985.