The influence of the ethanol concentration on the composition and activity of a developed biofilm in a denitrifying submerged unidirectional filter was studied. Process yields (represented as inorganic nitrogen removal), total platable bacteria, denitrifying bacteria, nitrate- and sulphate-reducing bacteria and denitrifying activity (N2O production) were compared at different ethanol concentrations (0 to 46.74 mg l(-1)). The biofilm exhibited a diverse bacterial composition and higher microbial development at the entrance of the unidirectional biofllter. The number of cells per gram of dry weight of biofilm was increased when the ethanol concentration increased, with the exception of nitrate reducers, for which the number of cells decreased per gram of biofilm in relation to height. Five different species of denitrifying bacteria were isolated from the biofilm, all of which were gram-negative rods. All of the species manifested an increase in denitrifying activity when the ethanol concentration was increased. In this sense, the number of denitrifying bacteria in the biofilm was positively correlated with the ethanol concentration. Both nitrate- and sulphate-reducing bacteria were present in the biofilm in the lower and higher part of the column. Nitrate-nitrogen removal in the submerged filter showed a high correlation with the influent ethanol concentration.