The isotropic and anisotropic hyperfine coupling constants and g-values of the nitroxide spin label (1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl)methanethiosulfonate (MTSSL) were determined from 9-GHz and 95GHz electron paramagnetic resonance (EPR) measurements in various solvents with a large distribution in polarity and proticity. The parameters A(iso), g(iso), A(zz), and g(xx) of MTSSL were found to be sensitive to changes in solvent properties, where A-values increased and g-values decreased due to increased solvent polarity or proticity. A linear correlation was found for the isotropic (g(iso), A(iso)) and anisotropic (g(xx), A(zz)) parameters, respectively. Furthermore, density functional theory (DFT) calculations of the same parameters were performed for a model spin label with the possibility to vary the dielectric constant (epsilon) of the medium and the number of hydrogen bonds formed with the nitroxide oxygen. From a qualitative analysis of experimental and calculated results, it was possible to specify the causes of the parameter shifts in more detail. In the "apolar region" (epsilon < 25), the sensitivity of A(iso) and A(zz) to epsilon is large. However, in the "polar region" (epsilon > 25), the sensitivity to epsilon is small, and the shifts in Ai,. and Azz are mainly determined by the proticity of the solvent. Methanol was found to form similar to1 and water similar to2 hydrogen bonds to the nitroxide on average. The DFT method determined the shifts in g(iso) and g(xx) due to hydrogen bonding more accurately compared with the restricted open-shell Hartree-Fock method. The anisotropic spin label-solvent data can be used in the interpretation of rigid-limit data from spin-labeled proteins to gain further insight in local environmental properties.