Transposon mutagenesis, although utilized extensively in cell-walled bacteria, has been used rarely within the class Mollicutes (1 ). The reasons for such limited use include a lack of the necessary techniques and transposons that contain suitable antibiotic markers. There has been limited success with methods of artificial transformation using the Gram-positive bacterial transposons Tn916 and Tn4001 . The most successful methods for transformation are a polyethylene glycol- (PEG) mediated procedure based on that used to transform Gram-positive bacterial protoplasts and electroporation. Species that have been transformed with these techniques include Acholeplasma laidlawii, Acholeplasma orale, Mycoplasma arthritidis, Mycoplasma pulmonis, Mycoplasma capricolum, Mycoplasma mycoides, Mycoplasma gallisepticum , and Spiroplasma citri (2 ,3 ). Transformation of mycoplasmas is a rather inefficient process usually requiring several micrograms of purified plasmid DNA to yield 10−6 –10−8 transformants/colony-forming unit (CFU) (3 ). Protocols for transforming mycoplasmas can be found in Chapter 25 .