The parameters affecting polyethylene glycol-induced plasmid transformation of Streptococcus lactis LM0230 protoplasts were examined to increase the transformation frequency. In contrast to spreading protoplasts over the surface of an agar medium, their incorporation into soft agar overlays enhanced regeneration of protoplasts and eliminated variability in transformation frequencies. Polyethylene glycol with a molecular weight of 3,350 at a final concentration of 22.5% yielded optimal transformation. A 20-min polyethylene glycol treatment of protoplasts in the presence of DNA was necessary for maximal transformation. The number of transformants recovered increased as the protoplast and DNA concentration increased over a range of 3.0 X 10(6) to 3.0 X 10(8) protoplasts and 0.25 to 4.0 micrograms of DNA per assay, respectively. With these parameters, transformation was increased to 5 X 10(3) to 4 X 10(4) transformants per microgram of DNA. Linear and recombinant plasmid DNA transformed, but at frequencies 10- to 100-fold lower than that of covalently closed circular DNA. Transformation of recombinant DNA molecules enabled the cloning of restriction endonuclease fragments coding for lactose metabolism into S. lactis LM0230 with the Streptococcus sanguis cloning vector, pGB301. These results demonstrated that the transformation frequency is sufficient to clone plasmid-coded genes which should prove useful for strain improvement of dairy starter cultures.