1. Four experiments, each with four concurrent continuous cultures of rumen micro-organisms, were used to investigate the effects of inoculum, food substrate and cobalt supplementation on the course of fermentation and vitamin B12 synthesis. The inocula came from sheep receiving either a Co-rich, complete diet (Ruminant A: Expts 1, 2 and 4) or a Co-deficient hay (Expt 3). In Expt 2, inocula from different donors were used for each culture but for other experiments they were pooled. Co-deficient hay was used as the initial substrate and Co-supplements were given after 8-54 d Co-depletion. 2. In three of four experiments, two using a pooled inoculum, uncharacteristically low acetate (Ac) and high propionate (Pr) outputs were obtained from the hay substrate in some cultures. In all, six cultures gave Ac:Pr values in the effluent less than 2:1 and, in Expt 3, the differences remained evident for at least 6 d after a change to a Co-deficient barley substrate. 3. Abnormal cultures gave lower cobalamin (cbl) and vitamin B12 analogue outputs than normal cultures: when supplemented with Co they showed small responses in cbl and large responses in analogue output with a slow increase in Ac:Pr. 4. We suggest that the use of Co-deficient substrates led to shifts in the microbial populations of some cultures, indicating the cbl-dependence of some species of rumen micro-organisms.