Transfection of Lactobacillus casei ATCC 27092 protoplasts with phage PL-1 DNA was studied under various conditions. The process of transfection was dependent on the incubation temperature, and the apparent activation energy was calculated to be about 11 kcal/mol. Transfection was inhibited by treating the cells before protoplasting either with monoiodoacetate, N,N'-dicyclohexylcarbodiimide (DCCD), or NaN3, without affecting both the viability of uninfected cells and protoplasting. The addition of DCCD after mixing protoplasts and DNA had no influence on transfection efficiencies. The transfection of L. casei protoplasts with phage PL-1 DNA was considered to require cell energy such as proton-motive force, probably in the initial stages, although the direct involvement of cell energy in the transfer of DNA across the cell membrane is still unclear.