The reaction of coenzyme B-12 (adenosylcobalamin) with cyanide has been reinvestigated in detail using spectroscopic and kinetic techniques. It has been shown that this reaction proceeds in one kinetically observable step, contradicting previous findings, with rate-determining attack of the first cyanide (k = (7.4 +/- 0.1) x 10(-3) M-1 s(-1), 25.0 degrees C, I = 1.0 M (NaClO4)). The activation parameters were found to be Delta H-double dagger = 53.0 +/- 0.6 kJ mol(-1), Delta S-double dagger = -127 +/- 3 J mol(-1) K-1 and Delta V-double dagger = -10.0 +/- 0.4 cm(3) mol(-1), suggesting an associative displacement mechanism. It is postulated that attack of the first cyanide occurs at the beta-(5’-deoxy-5’-adenosyl) site rather than at the alpha-dimethylbenzimidazole site.