The effects on membrane fluidity of inhibitors of acetylcholinesterase (A ChE) as well as those of certain nicotinic agonists have been examined in synaptosomes prepared from the electroplax of Torpedo californica. Electron paramagnetic resonance (EPR) spectrometry was used to monitor changes in the order parameters of three spin-labeled fatty acid probes, doxyl stearic acid derivatives, intercalated in the lipid bilayer of synaptosomal membranes. Neither inhibition of A ChE nor the application of nicotinic ligands significantly influenced the viscosity of the membrane bilayer as judged by changes in the order parameters of the probes. A small, narrow line component of the EPR spectra disappeared when high concentrations of acetylcholine (ACh) were added to the synaptosomes. This effect was not seen with other cholinergic agonists, e.g., car-bachol. The results of this investigation suggest that if anti-A ChEs produce damaging effects on the lipid components of nerve membranes, the effects are indirect, resulting from accumulation of high ACh levels and not from a direct action of the agent on the membrane lipid.