The solubilization site of two betalain pigments, namely, betanin and indicaxantin, into l-alpha-dipalmitoyl-phosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) vesicles was investigated by a spectrophotometric study. Pigment absorbance was monitored by varying phospholipid concentration, at a constant temperature that was varied in a range including the main phase transition temperature (Tm) of the relevant phospholipid bilayer. Maximum betanin absorption increased with the increase of DPPC concentration within the entire temperature range, reaching a plateau. The binding constant (Kb) of the pigment, calculated according to a pseudo-two-phase model, varied with the temperature, indicating that betanin is located at the hydrophobic interior of the bilayer. Other measurements of binding of betanin to DMPC and of indicaxanthin to either DPPC or DMPC vesicles ruled out that these compounds were solubilized in the hydrophobic interior of these bilayers.