The binding of lasalocid A to dipalmitoylphosphatidylcholine (DPPC) vesicles was studied following changes in the intrinsic fluorescence of this ionophore. The binding calculations indicated a dissociation constant of 6.98 +/- 1.5 muM at 48 degrees C, i.e., above the transition temperature (Tc) of the pure phospholipid, with a number of binding sites of 1 per 22 +/- 2.5 molecules of phospholipid, while at 23 degrees C, i.e., below the Tc of the pure phospholipid, the dissociation constant was 9.15 +/- 0.24 muM and the number of binding sites was 1 per each 29 +/- 1.6 molecules of DPPC. Changes in the temperature induced changes in fluorescence intensity of lasalocid A mainly upon phase changes, indicating a progressive decrease in the transition temperature accompanied by a broadening of the transition as lasalocid A concentration was increased. Fluorescence quenching experiments with N-methylnicotinamide showed a certain accessibility of the fluorophoric group of the ionophore to the aqueous quencher. Differential scanning calorimetry showed that increasing concentrations of lasalocid A drastically modified the thermotropic profile. At concentrations higher than 5 mol%, a second peak appeared, possibly due to a lateral phase segregation of lasalocid A trapping some phospholipid molecules. The results are interpreted in terms of limited solubility of lasalocid A in the phospholipid vesicles, this solubility being higher in fluid than in rigid phospholipid. Lateral segregation seems to occur with formation of more than one phase. At least the salicylic acid residue of the ionophore appears to be located near the polar head group of the phospholipid.