Spheroplasts were prepared from Corynebacterium glutamicum ATCC13032 by growing cells in the presence of glycine followed by digestion with lysozyme. Using pUL330 a spheroplast transformation system was established routinely yielding 103 to 104 transformants per μg of plasmid DNA. Spheroplasts were converted into protoplasts after incubation with the lytic enzyme achromopeptidase in the presence of additional lysozyme. Protoplasts prepared by this method regenerated at efficiencies of 10 to 30%. A protoplast transformation system was established routinely yielding 105 to 106 transformants per μg of plasmid DNA. The Escherichia coli Brevibacterium lactofermentum shuttle vector pUL62 prepared from E. coli could be introduced into spheroplasts of C. glutamicum after heat treatment at 48 to 49°C for 10 min.