Ferredoxin (Fd) is the soluble protein that accepts electrons from photosystem I (PSI) and makes them available to stromal enzymes in higher plant chloroplasts. In linear electron flow, Fd mainly donates electrons to Fd:NADPH reductase (FNR) which generates NADPH for use in the Calvin cycle, but Fd may also return electrons to the thylakoid plastoquinone pool, forming a cyclic electron flow. Many higher plants contain two different photosynthetic Fd proteins, but there are no conserved sequence differences that allow their division into evolutionary groups. In the model C3 photosynthesizing dicot, Arabidopsis thaliana, there are two such photosynthetic Fds, and we have exploited RNA interference (RNAi) techniques to specifically decrease transcript abundance of different Fds in this plant. Surprisingly, the perturbation of photosynthesis, as measured by cholorophyll fluorescence, in RNAi lines of the two different photosynthetic Fds shows opposite trends. Linear electron flow is retarded in lines with lower Fd2 (the most abundant Fd species) levels and under certain circumstances enhanced in lines with lower Fd1 (the minor isoprotein) levels. These data are evidences for at least partially differentiated roles of Fd1 and Fd2 in photosynthetic electron transfer, possibly in the partition of electrons into linear and cyclic electron flow.