This communication explicitly considers the possibility that local anesthetics interact with divalent cation binding centers, such as chlortetracycline, quin 2, ethyleneglycol bis (B-aminoethyl ether)-N-N,N',N'-tetraacetic acid (EGTA), ethylenediamine tetraacetic acid (EDTA) and ATP. Alterations of local anesthetic fluorescence spectra have been found in the presence of EGTA, EDTA and ATP. On the other hand, the fluorescence of chlortetracycline is enhanced and that of quin 2 is quenched by local anesthetics. The spectrofluorometric evidence presented in this paper clearly indicates that local anesthetics and these divalent cation chelators interact in solution. The fluorescence alterations observed do not derive from parallel changes of their respective absorption spectra, thus, they appear to be due to quantum yield changes. On the basis of the spectral perturbations observed, it is likely that local anesthetics interact with M2+ binding centers via their electron defective aromatic ring. From the association constants obtained in this study, we make an estimation of the free energy of this interaction ranging from -2.8 to -4.0 kcal/mole in the following experimental conditions: pH 7.4 at an ionic strength of 0.1 at 25 degrees. The relevance of these results to define the physical-chemical characteristics of the local anesthetic receptor site is briefly discussed. It is suggested that local anesthetics can bind strongly to Ca2+ and Mg2+ binding centers, provided that a hydrophobic region is located nearby.