Fluorine NMR paramagnetic relaxation enhancement was evaluated as a versatile approach for extracting distance information in selectively F-labeled proteins. Proof of concept and initial applications are presented for the HIV-inactivating lectin cyanovirin-N. Single F atoms were introduced at the 4-, 5-, 6- or 7 positions of Trp49 and the 4-position of Phe4, Phe54, and Phe80. The paramagnetic nitroxide spin label was attached to Cys residues that were placed into the protein at positions 50 or 52. (19) F-T2 NMR spectra with different relaxation delays were recorded and the transverse (19) F-PRE rate, (19) F-Γ2 , was used to determine the average distance between the F nucleus and the paramagnetic center. Our data show that experimental (19) F PRE-based distances correspond to 0.93 of the (1) HN -PRE distances, in perfect agreement with the gyromagnetic γ(19) F/γ(1) H ratio, thereby demonstrating that (19) F PREs are excellent alternative parameters for quantitative distance measurements in selectively F-labeled proteins.