Stable auxotrophic mutants of the methylotroph Methylophilus methylotrophus AS1 were obtained by a novel mutagenesis technique in which electroporation is used to transport the chemical mutagen N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) across the cell membrane. By combining chemical mutagenesis with electroporation and screening single colonies for auxotrophy in 36 different amino acids and growth factors, 3 auxotrophs per 156 colonies screened were obtained, whereas no auxotrophs were found with chemical mutagenesis alone. MNNG mutagen toxicity was also increased in the methylotroph with this novel mutagenesis technique (death rate 96% compared to 79%). This technique did not increase the mutation rate for strain Escherichia coli BK6 which responds well to simple exposure to the mutagen.