The strategy of betaine control for vitamin B-12 large-scale fermentation by Pseudomonas denitrificans was investigated in this paper. The results obtained in shake-flask experiments demonstrated that betaine could greatly stimulate vitamin B-12 biosynthesis but had an inhibition to cell growth. Based on the influence of betaine on the fermentation of P. denitrificans, betaine feeding was a beneficial strategy to solve the inconsistency between cell growth and vitamin B-12 production. As a result, an effective and economical strategy of betaine feeding was established for vitamin B-12 fermentation in 120-m(3) fermenter, in which betaine was continuously fed to maintain betaine concentration of the broth at the range of 5-7 g/l during 50-140 h of fermentation. (c) 2008 Published by Elsevier Ltd.