Fluorine-labeled analogues of Chromatium vinosum high-potential iron protein have been investigated by F-19 NMR spectroscopy. Fluorine-19 resonances have been assigned, and chemical shift variations, relaxation times, and temperature dependencies have been determined, Observed changes in fluorine chemical shifts and relaxation times following oxidation of the [Fe4S4] cofactor appear to reflect structural perturbation of the protein backbone and side chains, rather than variations in the paramagnetism of the cluster. Fluorine-19 NMR provides a probe of redox-dependent conformational change in electron-transfer proteins, which may also be of value for structural characterization of mutants. Evaluation of H2O/D2O solvent isotope effects on F-19 chemical shifts reflects solvent accessibility to various protein domains, while measurement of F-19 relaxation times affords a convenient test of the relative contribution of cross-relaxation to magnetization decay. For HiPIP, the results reported herein indicate that the cross-relaxation contribution to the longitudinal relaxation (T-1) is relatively small for both the oxidized and reduced states, Unusual temperature dependencies and fast relaxation times for the F-19 resonances of 3-F-Phe66 and 3-F-Tyr19 labeled HiPIP support a close interaction of these two residues with the iron-sulfur cluster.