Resonance Raman spectra of native 2[4Fe-4S] ferredoxin from Clostridium pasteurianum and of its selenium-substituted analogue 2[4Fe-4Se] are compared. The experimental conditions used in this study included low temperature (approx. 25K) and the absence of a glass sample cell, thus ensuring high signal-to-noise ratios. The spectra of the 2[4Fe-4S] and 2[4Fe-4Se] ferredoxins display similar numbers of bands, but the resonance Raman patterns differ largely, except for two bands observed at 353 cm-1 and 365 cm-1 in spectra of the native ferredoxin, which are only moderately shifted upon S----Se substitution and are attributed to Fe-S(cysteine) stretching modes. The activities of the latter modes, enhanced by 457.9 nm excitation, are nearly equal in both ferredoxins. These data were used to demonstrate the presence of hybrid clusters [4Fe-(4-n)S-nSe] (n = 1, 2, 3) in a ferredoxin the active sites of which had been reconstituted in the presence of both S and Se.