Aims: Nuclease secretion was evaluated for five species of Lactobacillus and the activity was characterized in terms of thermal resistance, molecular weight and mode of action on plasmid DNA. Methods and Results: Assays of nuclease from L. rhamnosus ATCC 9595 on DNA of different origins indicates a broad activity spectrum. Secreted nuclease from this strain resists a thermal treatment of 20 min at 100degreesC, is not sensitive to a treatment for disruption of disulphide bonds nor to EDTA treatment under 10 mmol l(-1). Nuclease production is not growth linked and seems to be constitutive. Extracellular nuclease of L. rhamnosus ATCC 9595 introduces a single-stranded nick in supercoiled DNA, thus potentially reducing the transformability of plasmid DNA. In seven of eight tested strains, SDS-PAGE revealed a major protein with a molecular weight of ca 35 kDa. Minor degradation products also showed nuclease activity. Conclusions: A comparative analysis of the extracellular fractions of 14 different Lactobacillus strains indicate that nuclease secretion seems to be a widely distributed function among species of milk-related lactobacilli. The production of secreted nuclease may contribute to the low ability of Lactobacillus spp. to be transformed and maintain exogenous DNA. Significance and Impact of the Study: Determination of the characteristics and distribution of nuclease activity contribute to developing strategies to overcome this barrier to efficient transformation of milk lactobacilli.