Ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii catalyzes the reduction of nucleotides to deoxynucleotides with concomitant oxidation of two cysteines within the active site to a disulfide. RTPR requires adenosylcobalamin (AdoCbl) as a cofactor and as a radical chain initiator. Catalysis is initiated by homolysis of the carbon-cobalt bond of AdoCbl to yield cob(II)alamin, 5'-deoxyadenosine, and a protein-based thiyl radical. The turnover numbers for ATP with its allosteric effector dGTP, and for CTP with its allosteric effector dATP, are both 2 s(-1). The rate-limiting step for turnover in the steady state is re-reduction of the oxidized form of the protein, a conformational change, or both. Under conditions where [RTPR] much greater than [AdoCbl], the rates of ATP and CTP reduction do not vary linearly with [AdoCbl] but instead exhibit saturation behavior with turnover numbers of 10 s(-1) (ATP) and 8.5 s(-1) (CTP). This result suggests that dissociation of AdoCbl, which requires carbon-cobalt bond reformation, follows nucleotide reduction, but precedes the rate-limiting step in catalysis. A presteady-state analysis of the ATP reduction (using rapid chemical quench methods) in the presence of [5'-H-3]-AdoCbl reveals formation of product dATP at a k(obs) of 55 +/- 10 s(-1) and tritium washout from [5'-H-3]-AdoCbl at 0.6 s(-1). The rate of washout is approximately equivalent to the rate of washout of H-3 in the absence of substrate. Measurement of the ratio of (H2O)-H-3:dATP over time reveals that washout of H-3 occurs at the end of each turnover. Production of (H2O)-H-3 requires reformation of the carbon-cobalt bond. These steady-state and presteady-state data suggest that carbon-cobalt bond reformation and dissociation of AdoCbl into solution accompany each turnover and that the radical chain length of the RTPR-catalyzed nucleotide reduction is approximately one.