A mixed bacterial culture, isolated from a petroleum-contaminated site, was evaluated for its de-emulsification capabilities using a surfactant-stabilized kerosene-water model system. The culture produced high de-emulsification activity with an initial de-emulsification rate of 44%/h (44% of emulsion separated in 1 h) and a maximum extent of de-emulsification of 96% in 24 h. A linear relationship was observed between the initial rate of de-emulsification and culture concentration and a logarithmic relationship was observed between the extent of de-emulsification and culture concentration. The de-emulsification activity of the mixed bacterial culture was mainly associated with the microbial cells. Culture grown on petroleum hydrocarbon substrate exhibited higher de-emulsification capability as compared with the carbohydrate substrate grown culture. The culture's ability to break emulsions was not significantly affected by lyophilization or freezing and thawing, but was completely destroyed by autoclaving. The mixed culture effectively de-emulsified various water-in-oil field emulsions. (C) 2002 Elsevier Science Ltd. All rights reserved.