In the present study, water-soluble nonenzymatic browning products (melanoidins) formed in roasted malt were separated, quantified, and investigated for their effects on detoxifying mechanisms in intestinal Caco-2 cells. The melanoidins were prepared from roasted malt by hot water extraction, and the water-soluble compounds were separated into different molecular weight (MW) fractions by gel filtration chromatography. By monitoring the effluent at 300 nm, seven molecular fractions I-VII were consecutively collected, revealing that approximately 2.3% of the water-soluble compounds had mean MWs between 10000 and 30000 Da. Thus, the bulk of water-soluble malt melanoidins consisted of MW > 30000 Da, among which approximately 58% showed mean MWs between 60000 Da and 100000 Da, whereas approximately 32% exhibited mean MWs of 200000 Da. Biotransformation enzyme activities of NADPH-cytochrome c-reductase (CCR) and glutathione-S-transferase (GST) were analyzed in Caco-2 Cells after 48 h of exposure to the different MW fractions. The low MW fraction of 10000 Da was most effective in activating the CCR and the GST activities (+122 and +33% vs control, respectively). The majority of the mid molecular weight compounds tested showed an activating effect on CCR activity and an inhibitory effect on GST activity. These effects were most pronounced for compounds of up to 70000 Da and >200000 Da but less distinct for fractions of an average molecular weight of 100000 Da.