Electron paramagnetic resonance (EPR) spin-labeling methods were used to study the effects of carotenoids on the physical properties of saturated phosphatidylcholine (PC) membranes to evaluate the contribution of the terminal hydroxyl groups of xanthophyll molecules to the carotenoid-membrane interaction. Effects of the dipolar, terminally dihydroxylated carotenoid lutein on membrane phase transition, fluidity, order, and polarity were compared with those of monopolar (beta-cryptoxanthin) and nonpolar (beta-carotene) carotenoids. These effects were monitored at the membrane center as a function of the amount of the carotenoid added to the sample and as a function of temperature for fluid-phase membranes. PC membranes with different thickness (from 12 to 22 carbons in alkyl chains) were used. Carotenoids shifted to lower temperatures and broadened the main phase transition of PC membranes. They decreased the membrane fluidity and increased the order of alkyl chains. Carotenoids also increased the hydrophobicity of the membrane interior. These effects were the strongest for lutein, significantly weaker for beta-cryptoxanthin, and negligible for beta-carotene. They decreased with the increase of the membrane thickness. Presented results suggest that anchoring of carotenoid molecules at the opposite membrane surfaces by polar hydroxyl groups is significant in enhancing their effects on membrane properties. This manuscript also shows the ability of EPR spin-labeling methods to monitor different membrane properties that can be applied in biotechnological studies with the use of liposomes.