The anaerobic Escherichia coli ribonucleotide reductase (class III reductase) responsible for the synthesis of the deoxyribonucleotides required for anaerobic DNA replication contains an oxygen-sensitive glycyl radical (Gly-681) suggesting involvement of radical chemistry in catalysis. The amino acid sequence of this enzyme completely differs from that of earlier described aerobic class I (prototype, aerobic E. coli) and class II (prototype, Lactobacillus leichmanii) reductases that use radical chemistry but employ other means for radical generation. Here, we study the interaction between the anaerobic E. coli reductase with the 5'-triphosphates of 2'-chloro-2'-deoxycytidine, 2'-fluoro-2'-deoxycytidine, and 2'-azido-2'-deoxycytidine (N3CTP), which are mechanism-based inhibitors of class I and II reductases and, on interaction with these enzymes, decompose to base, inorganic di(tri)phosphate and 2'-methylene-3(2H)-furanone. Also, with the anaerobic E. coli reductase, the 2'-substituted nucleotides act as mechanism-based inhibitors and decompose. N3CTP scavenges the glycyl radical of the enzyme similar to the interaction of N3CDP with the tyrosyl radical of class I enzymes. However, we found no evidence for a new transient radical species as is the case with class I enzymes. Our results suggest that the chemistry at the nucleotide level for the reduction of ribose by class III enzymes is similar to the chemistry employed by class I and II enzymes.