Conditions for protoplast formation and cell wall regeneration of Lactobacillus casei YIT 9018 were studied. L. casei YIT 9018 was converted to protoplast easily by treating with N-acetylmuramidase(mutanolysin) in 20mM potassium phosphate buffer(pH 6.8) containing 6mM CaCl₂, 6mM MgCl₂ and IM sucrose. The optimum conditions of protoplast formation were obtained when cells were taken from TCM broth containing 0.5% glycine at middle or late logarithmic growth phase. Cells were regenerated on a TCM medium supplemented with 6mM CaCl₂, 6mM MgCl₂, 0.8M sucrose and 0.5% bovine serum albumin. The regeneration frequency of protoplasts to normal cells was 3.30% at 30℃.