A new high-potential iron-sulfur protein (HiPIP) has been isolated and purified to homogeneity from the soluble fraction obtained from light-grown cells of the facultative photoheterotrophic bacterium Rhodoferax fermentans. The new protein was identified as a HiPIP by virtue of its molecular properties such as the molecular mass (Mr = 8.7 kDa), the Fe/protein ratio (3.8 ± 0.2), the reduction potential (Em,7 = +351 mV), the electronic spectrum of the reduced and the oxidized protein, and the EPR spectrum of the oxidized protein. These molecular properties lie in the range observed for HiPIPs from other sources and, in particular, the iron content is consistent with the presence of one [Fe4S4] cubane cluster per molecule. The isoelectric pH values of the two redox forms are consistent with a basic protein. Kinetic studies of HiPIP oxidation, performed by monitoring the absorbance changes induced upon light excitation of the photosynthetic reaction center, give direct evidence of the role of the HiPIP in the photosynthetic electron transfer chain of Rf. fermentans.