Various factors affecting protoplast formation in lactobacilli were investigated. First, we studied growth conditions and found that mid- to latelogarithmic growth phase cells were most effectively converted to protoplasts with lytic enzyme (s), and that cells grown on glucose-limited broth showed the high efficiency in protoplast formation regardless of the growth phase. Secondly, reaction conditions were tested. The efficiency of protoplasting depended greatly on osmotic stabilizers. We checked about 50 chemicals for suitable stabilizers and discovered that citrate was very effective. We found also that the autolytic activity of the cells was always high under optimum growth and reaction conditions for protoplasting. From these and other results, we speculate that protoplast formation in lactobacilli is achieved by mutual action of both foreign lytic enzyme(s) and autolytic enzyme(s).