The effects of halothane, thiopental, and lidocaine were studied with spin-labeling methods in synaptic plasma membranes (order parameter) and artificial phospholipid membranes (lateral diffusion). Halothane had a biphasic action, low concentrations (0.64 mM) ordering and high concentrations (2.9 mM) fluidizing both types of membranes. A biphasic effect in phospholipid membranes was also seen with thiopental, 0.1 mM ordering and 10 mM fluidizing, whereas in synaptic plasma membranes both low and high concentrations caused an increased order in the lipid bilayer region. At high thiopental concentrations, a considerable number of molecules may have reacted with membrane proteins or accumulated in the highly fluidic hydrophobic interior region of the membrane without affecting the rotational movement of the labeled fatty acid. Lidocaine alone, or together with calcium chloride, at various concentrations to 10 mM had no significant effect, and a fluidizing effect of 1 mM calcium chloride was possibly a result of interaction of calcium chloride with the label. The results indicate that the three lipid-soluble anesthetics interact differently with the lipid part of membranes. Lidocaine did not seem to affect bilyer lipids, while thiopental and halothane in phospholipid vesicles and halothane alone in synaptic membranes caused a dose-dependent biphasic effect.