The optimal conditions for the production and regeneration of L. helveticus protoplasts were examined. The protoplast formation of L. helveticus was most efficient obtained when the cells grown to mid and late logarithmic phase in MRS medium were used. The maximum number of protoplasts was obtained when lysozyme and mutanolysin were used to lysis the cell wall in 20mM HEPES buffer (pH 7.0) containing 1M sucrose. Regeneration was accomplished with a complex medium containing 10% sucrose, 10mM MaCl2, 20mM CaCl2, 5% gelatin and 0.5% bovine serum albumin. The regeneration frequency of the protoplasts was 10-20% after 5 days of incubation at 30C.