The utilisation of lactose, lactulose and their mixtures in fermentation by the strain Lactobacillus acidophilus CCDM 92 was followed. Experiments were carried out in vitro under standard conditions (MRS agar, 1 vol.% of inoculum, cultivation for 16 h at 37 °C). In the course of fermentation, lactose, lactulose and lactic acid (HPLC), the count of bacteria (classic plate method) and pH were monitored in substrates. The present results demonstrated: (i) different courses of saccharide utilization and lactic acid production in the fermentation of substrates with addition of lactose or lactulose; (ii) the preference of predominant saccharide fermentation in substrates with lactose and lactulose mixtures (6:1 and 1:6); (iii) considerable reduction of lactulose consumption during the fermentation of substrate containing a lactose and lactulose mixture (1:1). The reason is the feed-back inhibition effect of lactic acid cumulated in the substrate. The findings support the hypothesis that under the test conditions, the lactulose metabolism is not linked to the known lactose utilization pathways.