The effects of local anesthetics on the topology of aminophospholipids and on the release and uptake of dopamine in rat brain synaptosomes have been examined. A metabolically intact preparation of synaptosomes was prepared which maintains amino-phospholipid asymmetry and the capacity for sodium-driven uptake and depolarization-dependent release of dopamine. Incubation of synaptosomes with local anesthetics at 37 degrees C induced perturbations in the topology of aminophospholipids as determined by their reactivities to the covalent probe trinitrobenzenesulfonic acid. The reaction of trinitrobenzenesulfonate with phosphatidylethanolamine and phosphatidylserine was inhibited 10-20% by low concentrations of tetracaine (1-100 muM) and enhanced by high concentrations (0.3-1.0 mM). Other local anesthetics showed a similar biphasic effect with a potency order of dibucaine greater than tetracaine greater than lidocaine greater than or equal to procaine. K+-stimulated, Ca2+-dependent release of [3H]dopamine was inhibited significantly at low concentrations of tetracaine (1-10 muM) but enhanced at higher concentrations (0.1-1.0 mM). Dibucaine and procaine had a similar biphasic effect on the dopamine release. For each of the local anesthetics tested, the inhibition of the reaction of phosphatidylethanolamine and phosphatidylserine with trinitrobenzenesulfonate occurred at concentrations which were shown also to inhibit the release of [3H]dopamine. Local anesthetics were shown to inhibit uptake of [3H )dopamine with a potency order which reflects their potency in producing anesthesia. The inhibition of dopamine uptake by dibucaine, tetracaine, lidocaine, or procaine was characterized by inhibitory constants (KI) of 1.8 +/- 0.4 muM, 27 +/- 5 muM, 190 muM and 0.5 mM, respectively.