This chapter describes the partial purification of ribosome-associated vitamin B 12 adenosylating enzyme of Lactobacillus leichrnannii and the properties of the enzyme. Adenosylcobalamin (AdoCbl) synthesized in an initial incubation containing adenosylating enzyme, vitamin B 12r , ATP, and MnCl 2 is assayed in a second incubation by means of an AdoCbl-dependent enzyme reaction, the rate behavior of which is a function of AdoCbl concentration. The initial incubation of the adenosylating enzyme assay is conducted in small Thunberg tubes initially, containing the ingredients: cyanocobalamin (or hydroxocobalamin), 15 nmol; ATP, 0.088/μmol; MnCI 2 , 0.13/μmol; and Tris–HC1, pH 8.0, 20 μmol, in a total volume of 0.4 ml. Tubes are placed in ice, evacuated, and flushed with nitrogen. Potassium borohydride, 1 mg, is added to each tube under a stream of nitrogen, followed by enzyme-containing samples. Tubes are again evacuated, flushed with nitrogen, and kept chilled until reduction of vitamin B 12a is complete. The tube is incubated for 45 min in the dark at 37°, then cooled to 0°, a 10- to 50-μl aliquot is assayed for AdoCbl. Ribosomes from large-scale cultures of L. leichmannii are washed in TSM buffer and stored at –20° until use. All purification steps are performed at 0–4°.