Tetracaine-induced biphasic structure-functional alterations were investigated in acetylcholinesterase (AChE)-associated membrane vesicles from the electric organ of Torpedo californica. Enzyme assays showed that tetracaine exhibits a biphasic effect on the activity of membrane-bound AChE: increasing it at low concentrations (< 12 mM) and decreasing it at high concentrations (> 12 mM). Fluorescence-polarization experiments demonstrated that tetracaine affects the fluidity of lipid hydrocarbon chains of these membranes in a biphasic manner: increasing it at < 20 mM and decreasing it at > 20 mM. This small molecule also alters the fluidity of the negatively charged lipid head group: increasing it at < 13 mM and remaining essentially at the same level at > 13 mM. The positively charged lipid head group is unaffected. Contrasting effects on AChE activity with changes in membrane fluidity showed that [tetracaine] for AChE activity is comparable to that for the fluidity of the negatively charged lipid head group (12 mM versus 13 mM), but lower than that for a biphasic effect on the fluidity of lipid hydrocarbon chains (12 mM versus 20 mM). Differential scanning microcalorimetry showed that, due to membrane protein-lipid interaction, the lipid-phase transition temperature (tml) is higher for AChE-associated membrane vesicles than for isolated lipids from these membranes. An overall disordering of the membranes by tetracaine, as inferred from the lowering of tml, was also demonstrated. These findings suggested that binding of tetracaine to the lipid polar head group and membrane protein-lipid interaction may contribute to a higher [tetracaine] in inducing a comparable biphasic effect on membrane fluidity. At high [tetracaine], charge interactions between the tetracaine cation and the negatively charged lipid head group may result in a new lipid phase in the membranes, which could reverse the increase in membrane fluidity, resulting in the observed biphasic effect. Although both tetracaine and alcohol are amphiphilic species, they exhibit distinctive structure-functional effects on the membranes, as shown by comparing the results obtained on tetracaine with those previously reported for alcohol. The present observations may have significant physiological implications and may be of importance in understanding the biochemical effects of tetracaine in correlation with its physiological impact.