Protoplasts of L. plantarum were produced by mutanolysin-lysozyme digestion at 50°C and regenerated at a frequency of 1.6 to 3.8%. The addition of Tween 80 to the growth medium increased the length of time required for protoplast formation. When transfected with bacteriophage B2 DNA, transfection efficiencies ranged from 25 to 230 PFU/,ug of DNA and from 2.2 x 10-5 to 4.7 x 10-4 PFU per recovered protoplast. Total transfectant yield was 3.7 x 102 to 3.4 x 103 per treatment. Transformations with plasmid DNA were unsuccessful.