The reaction between coenzyme B-12 (5'-deoxyadenosylcobalamin, AdoCbl) and tetrabutylammonium cyanide to give dicyanocobalamin, adenine, and 1-cyano-D-erythro-2,3-dihydroxy-4-pentenol has been examined in 92% N,N-dimethylformamide (DMF)/8% D2O. Under these conditions rate-determining Co-C heterolytic cleavage is preceded by rapid addition of cyanide to AdoCbl to form an intermediate, (beta -5'-deoxyadenosyl)(alpha -cyano)cobalamin ((beta -Ado)(alpha -CN)Cbl(-)), identified by H-1 NMR spectroscopy. Rate constants have been determined by both H-1 NMR and visible spectroscopies, with the latter showing saturation kinetics. The observed rate constant is pH-independent in the pH region studied, and replacing D2O by H2O increases it by ca. 10%. Increasing the percentage of D2O in the DMF/D2O solvent mixture also increases the reaction rate, and for D2O greater than or equal to 50% there is a change in the rate-determining step, with formation of the (beta -Ado)(alpha -CN)Cbl(-) intermediate becoming rate-determining. A mechanism in 92% DMF/8% D2O is proposed which involves rapid reversible formation of (beta -Ado)(alpha -CN)Cbl(-) from base-off AdoCbl plus cyanide, followed by rate-determining solvent-assisted cleavage of the Co-C bond of the intermediate and subsequent rapid addition of a second cyanide to give the products.