The synthesis and characterization of spin-labeled phospholipids (SLP)-derivatives of 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol (PTE)-with pH-reporting nitroxides that are covalently attached to the lipid's polar headgroup are being reported. Two lipids were synthesized by reactions of PTE with thiol-specific, pH-sensitive methanethiosulfonate spin labels methanethiosulfonic acid S-(1-oxyl-2,2,3,5,5-pentamethylimidazolidin-4-ylmethyl) ester (IMTSL) and S-4-(4-(dimethylamino)-2-ethyl-5,5-dimethyl-1-oxyl-2,5-dihydro-1H-imidaz ol-2-yl)benzyl methanethiosulfonate (IKMTSL). The pK(a) values of the IMTSL-PTE lipid measured by EPR titration in aqueous buffer/isopropyl alcohol solutions of various compositions were found to be essentially the same (pK(a) approximate to 2.35), indicating that in mixed aqueous/organic solvents, the amphiphilic lipid molecules could be shielded from changing bulk conditions by a local shell of solvent molecules. To overcome this problem, the spin-labeled lipids were modeled by synthesizing IMTSL- and IKMTSL-2-mercaptoethanol adducts. These model compounds yielded the intrinsic pK(a)(0)'s for IMTSL-PTE and IKMTSL-PTE in aqueous buffers as 3.33 +/- 0.03 and 5.98 +/- 0.03, respectively. A series of EPR titrations of IMTSL-PTE in mixed water/isopropyl alcohol solution allowed for calibrating the polarity-induced pK(a) shifts, Delta pK(a)(Pol), vs bulk solvent dielectric permittivity. These calibration data allowed for estimating the local dielectric constant, epsilon(eff), experienced by the reporter nitroxide of the IMTSL-PTE lipid incorporated into the nonionic Triton X-100 micelles as 60 +/- 5 and 57 +/- 5 at 23 and 48 degrees C, respectively. For micelles formed from an anionic surfactant sodium dodecyl sulfate (SDS) the electrostatic-induced pKa shift, Delta pK(a)(el) = 2.06 +/- 0.04 units of pH, was obtained by subtracting the polarity-induced contribution. This shift yields Psi = -121 mV electric potential of the SDS micelle surface.