Fluorescent probe techniques were used to evaluate the effect of dibucaine.HCl on the physical properties (transbilayer asymmetric lateral mobility, annular lipid fluidity and protein distribution) of synaptosomal plasma membrane vesicles (SPMV) isolated from bovine cerebral cortex. An experimental procedure was used based on selective quenching of 1,3-di(l-pyrenyl)propane (Py-3-Py) by trinitrophenyl groups, and radiationless energy transfer from the tryptophans of membrane proteins to Py-3-Py. Dibucaine.HCl increased the bulk lateral mobility, and annular lipid fluidity in SPMV lipid bilayers, and had a greater fluidizing effect on the inner monolayer than the outer monolayer. The magnitude of increasing effect on annular lipid fluidity in SPMV lipid bilayer induced by dibucaine.HCl was significantly far greater than magnitude of increasing effect of the drug on the lateral mobility of bulk SPMV lipid bilayer. It also caused membrane proteins to cluster. These effects of dibucaine.HCl on neuronal membranes may be responsible for some, though not all, of the local anesthetic actions of dibucaine.HCl.