Although it is agreed that the bilayer lipid region of neural membranes is a principle site of action of general anesthetics, the nature of the action is somewhat controversial, and may depend on the structure of the membranes and the concentration of the anesthetics. Spin-labeled rat brain myelin membranes were shown to have higher order in their bilayer lipid region than that of synaptic plasma membranes, which was in turn higher than that of synaptic mitochondrial membranes, and corresponded to their respective contents of cholesterol (highest in myelin, lowest in mitochondria). Two broad discontinuities in the temperature dependence of the order parameter were observed in the bilayer lipids of all three membranes, a lower one between 12 and 20°C and a higher one between 32 and 38°C. Low concentrations of halothane (0.25 and 0.5 mM) and lidocaine (0.1 mM) increased the order in synaptic plasma membranes, but did not affect myelin or mitochondria membranes in the temperature range between 32 and 42°C. In synaptic plasma membranes the ordering effect of halothane switched to a fluidizing one between anesthetic concentrations of 0.75 and 1.1 mM. High concentrations (2.3 mM halothane and 10 mM lidocaine) decreased the order (fluidized) in all three membranes and was observed most clearly in the hydrophobic region of the lipid bilayer. 5-Hydroxytryptamine (5-HT) at 10−2 and 10−1 mM increased the order in synaptic plasma membranes and at some temperatures also in synaptic mitochondrial membranes, but not in myelin membranes. 5-HT antagonized the fluidizing effect of 2.3 mM halothane and 10 mM lidocaine in synaptic plasma membranes. The antagonism seemed to be only partial in the hydrophilic region of the bilayer lipids in mitochondrial membranes, but total in the hydrophobic region by 10−1 mM 5-HT. Although relatively high concentrations of 5-HT were used, the results showed that this neurotransmitter is able to influence the bilayer lipid fluidity particularly in synaptic plasma membranes. The results, furthermore, indicate that the biphasic dose-dependent effect on lipid fluidity of halothane and lidocaine occurs only in synaptic plasma membranes, and it is suggested that the difference between the degree of fluidity and the responses in the membranes investigated are due to differences in lipid compositions.