To provide a basis for studying the pharmacological actions of tetracaine HCl, we analyzed the membrane activities of this local anesthetic. The n-(9-anthroyloxy) stearic and palmitic acid (n-AS) probes (n = 2, 6, 9, 12 and 16) have been used previously to examine fluorescence polarization gradients. These probes can report the environment at a graded series of depths from the surface to the center of the membrane bilayer structure. In a dosedependent manner, tetracaine HCl decreased the anisotropies of 6-AS, 9-AS, 12-AS and 16-AP in the hydrocarbon interior of synaptosomal plasma membrane vesicles isolated from bovine cerebral cortex (SPMV), and liposomes derived from total lipids (SPMVTL) and phospholipids (SPMVPL) extracted from the SPMV. However, this compound increased the anisotropy of 2-AS at the membrane interface. The magnitude of the membrane rotational mobility reflects the carbon atom numbers of the phospholipids comprising SPMV, SPMVTL and SPMVPL and was in the order of the 16, 12, 9, 6, and 2 positions of the aliphatic chains. The sensitivity of the effects of tetracaine HCl on the rotational mobility of the hydrocarbon interior or surface region was dependent on the carbon atom numbers in the descending order 16-AP, 12-AS, 9-AS, 6-AS and 2-AS and on whether neuronal or model membranes were involved in the descending order SPMV, SPMVPL and SPMVTL.