The coenzyme F420 content of granular sludge grown on various substrates and substrate combinations was measured, and the potential of the sludge to form methane (maximum specific methane production rate) from hydrogen, formate, acetate, propionate, and ethanol was determined. The F420 content varied between 55 nmol g of volatile suspended solids (VSS)-1 for sludge grown on acetate and 796 nmol g of VSS-1 for sludge grown on propionate. The best correlation was found between the F420 content and the potential activity for methane formation from formate; almost no correlation, however, was found with acetate as the test substrate. The ratio between the potential methanogenic activities (qCH4) of sludges grown on various substrates and their F420 content was in general highest for formate (48.2 μmol of CH4 μmol of F420-1 min-1) and lowest for propionate (6.9 μmol of CH4 μmol of F420-1 min-1) as test substrates. However, acetate-grown granular sludge with acetate as test substrate showed the highest ratio, namely, 229 μmol of CH4 μmol of F420-1 min-1. The data presented indicate that the F420 content of methanogenic consortia can be misleading for the assessment of their potential acetoclastic methanogenic activity.