.The currently presumed assignment of CO/CN ligands in the structure of the active cluster in CO-inactivated [FeFe] hydrogenase is shown to be inconsistent with the available IR data in the enzyme from Clostridium pasteurianum I. A different arrangement has the correct qualitative and quantitative features, reproducing the observed line spacing and intensities and the observed line shift consequent to inactivation with labeled (CO)-C-13 instead of (CO)-C-12. The new assignment is also consistent with the observed change from rhombic to axial symmetry of the electron paramagnetic resonance g tensor upon inactivation.