Ribonucleotide reductase (RNR) from Lactobacillus leichmannii, a 76 kDa monomer using adenosylcobalamin (AdoCbl) as a cofactor, catalyzes the conversion of nucleoside triphosphates to deoxynucleotides and is rapidly, ( < 30 s) by I equiv of 2',2'-difluoro-2'-deoxycytidine 5'-triphosphate (F2CTP). [1'-H-3]- and [5-H-3]F2CTP were synthesized and used independently to inactivate RNR. Sephadex G-50 chromatography of the inactivation mixture revealed that 0.47 equiv of a sugar was covalently bound to RNR and that 0.71 equiv of cytosine was released. Alternatively, analysis of the inactivated RNR by SDS-PAGE without boiling resulted in 33% of RNR migrating as a 110 kDa protein. Inactivation of RNR with a mixture of [1'-H-3]F2CTP and [1'-H-2]F2CTP followed by reduction with NaBH4, alkyl, iodoacetamide, trypsin digestion, and HPLC separation of the resulting peptides allowed isolation and identification by MALDI-TOF mass spectrometry (MS) of a H-3/H-2-labeled peptide containing C-731 and C-736 from the C-terminus of RNR accounting for 10% of the labeled protein. The MS analysis also revealed that the two cysteines were cross-linked to a furanone species derived from the sugar of F2CTP. Incubation of [1'-H-3]F2CTP with C119S-RNR resulted in 0.3 equiv Of Sugar being covalently bound to the protein, and incubation with NaBH4 subsequent to inactivation resulted in trapping of 2'-fluoro-2'-deoxycytidine. These studies and the ones in the preceding paper (DOI: 10.1021/bi9021318) allow proposal of a mechanism of inactivation of RNR by F2CTP involving multiple reaction pathways. The proposed mechanisms share many common features with F2CDP inactivation of the class I RNRs.