Reduction of the first known halogen-containing substrate by nitrogenase (N(2)ase), 3,3-difluorocyclopropene (DFCP), was investigated. Reduction requires both N(2)ase proteins (MoFe and Fe protein), ATP, and an exogenous reductant (dithionite, DT), as with N-2 and known alternative substrates of the enzyme. Two major products providing evidence for reductive C-F bond cleavage were confirmed, propene (P1, requiring 6e(-)/6H(+)) and 2-fluoropropene (P2, 4e(-)/4H(+)). Both were identified by GC-MS and NMR spectroscopy, and had the same K-m constants (0.022 atm, 5.4 mM). Reduction of 1,2-dideuterated DFCP (d(2)-DFCP) further revealed that (i) in both PI and P2, two deuterium atoms are retained, one on carbon-1 and one on carbon-3, indicating that C=C bond cleavage rather than C C bond cleavage is involved during DFCP reduction at least to P2 (assuming no F migration); (ii) no selectivity was observed in formation of cis and trans isomers of 1,3-d(2)-2-fluoropropene, whereas cis-1,3-d(2)-propene is the predominant 1,3-d(2)-propene product, indicating that one of the bound reduction intermediates on the pathway to propene is constrained geometrically. A reduction mechanism, consistent with hydride transfer as a key step, is discussed. Reductive C-F bond cleavage is an ability of N(2)ase that further demonstrates the unique and remarkable scope of its catalytic prowess.