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Three different techniques for bacterial mating were applied to wild type and culture collection strains of Lactococcus lactis harbouring transposons: direct plate conjugation, filter mating and mating on milk agar. Efficiencies and frequencies of transfer were compared. Transconjugants were characterized by marker properties and molecular assays. Transposon-coded Suc+Nis+ phenotype as well as Suc+Bac+Nis− phenotype were transferred with frequencies ranging between 10−9 and 10−6. Milk agar plate mating was the best technique for obtaining gene transfer events involving wild type lactococci.