A new, simple procedure for the determination of partition coefficients (P) was developed based on spectral effects caused upon addition of solutes to spin labeled model lipid membranes, and on the knowledge of their water solubility. Values of P were determined for nine local anesthetics (LA), amino-esters and amino-amides. The results were in good agreement with those found by phase separation and by a more complex, previously reported, methodology (Lissi et al. (1990) Biochim. Biophys. Acta 1021, 46-50) applied to either EPR or fluorescence spectra of probes incorporated in the bilayers. Both the present and the previously reported procedures make use of effects on membrane structure evaluated by spectroscopic techniques and offer the advantage of not requiring phase separation. The spectral effects, indicative of a decrease in bilayer organization increased with LA concentration, reaching a maximum at the drug water solubility, indicating that partitioning in the membrane is limited by saturation of the aqueous phase. A thermodynamic analysis of the partition data according to Hill (Hill, M.W. (1974) Biochim. Biophys. Acta 356, 117-124) showed that the LAs did not display ideal behavior. Knowledge of the partition coefficients allowed a comparison between effects at the same drug concentration in the membrane. Within a given family (esters, acyclic amides, cyclic amides) no clear proportionality was observed between effect and LA hydrophobicity, as reflected in the partition coefficient. Rather, the membrane perturbing ability is a result of steric effects originating in the mismatch between anesthetic and phospholipid shapes.