**1. Introduction**

Lipid spin labels containing nitroxide groups at different positions in the fatty acid chain, such as 1-palmitoyl-2-stearoyl-(*n*-doxyl)-*sn*-glycero-3-phosphocholines (*n*-PC spin labels) are a useful and proven tool in lipid research. They have provided important insights into the structure of model and biological membranes, reported on the membrane fluidity, polarity, phase state and presence of microscopic domains1, accessibility of different depth positions in the lipid bilayer for oxygen and other polar and non-polar paramagnetic compounds2-4 and protein/lipid interactions 5, 6.

It is generally accepted, that, unlike bulky fluorescent labels7,8, nitroxides are well incorporated into fluid lipid bilayers9 and not excluded from them. However, it has been shown by NMR that although the most probable location of the nitroxide group for 5-, 10 and 16- PC spin labels in the fluid POPC membrane corresponds to the fully extended conformation, the distribution is relatively broad and other conformations should also be present10. Bent conformations were previously found for doxylstearic acids in monomolecular films11, water/hydrocarbon emulsion particles12 and micellar systems13. In fluid membranes the fluidity, polarity and accessibility parameters reported by ESR using PC spin labels and *n*-doxylstearic acids are, in general, change monotonically with an increase in *n*3, 14, although there are indications that the spin label groups on the stearates are located nearer to the membrane exterior than the analogous positions of the unlabeled phospholipid chains15. However, in the gel phase, which is characterized by denser chain packing and higher order, the preferential location may be different.

In this chapter we focus on the behavior of PC spin labels in the gel phase and frozen membranes. We show how the superior *g*-factor resolution of HF ESR provides new insights

in this behavior and a new look at the vast body of experimental data accumulated with PC spin labels in the last 30 years. In particular, we revisit so-called "polarity profiles" determined from the *g*-factor values and hyperfine splittings of PC spin labels in frozen phospholipid membranes with or without cholesterol and show that these values are affected by a number of factors in the membrane composition, chain packing in the lipid phase and folding properties of the *sn-2* spin labeled chain PC labels rather than reflect gradients of polarity or water content present in the membrane16.
