To evaluate the possible role of glutathionylcobalamin (GS-Cbl) in the intracellular metabolism of cobalamin, the following reactions were analyzed using extracts of rabbit spleen: (i) decyanation of cyanocobalamin; (ii) utilization of GS-Cbl by cobalamin reductase; (iii) participation of GS-Cbl in methionine biosynthesis; and (iv) conversion of GS-Cbl to adenosylcobalamin. Decyanation of cyanocobalamin required reduced glutathione which appeared to form a complex with the cobalamin. This complex decomposed during the extraction steps to sulfitocobalamin which was identified by high-performance liquid chromatography. Cobalamin reductase in spleen extract was more active with GS-Cbl than with aquocobalamin or cyanocobalamin as substrates (specific activities: 10.4, 2.8 and 0.93 nmol/mg/min, respectively). Methionine synthase utilized GS-Cbl as cofactor more efficiently than aquocobalamin or cyanocobalamin based on initial rates of enzyme activity. This suggests that GS-Cbl is a more direct precursor of the coenzyme required for methionine synthase. Formation of adenosylcobalaminm from GS-Cb1 was four times greater than from aquocobalamin alone. Based on these results, we propose that GS-Cbl or a closely related thiol-cobalamin adduct is a proximal precursor in cobalamin coenzyme biosynthesis.