Azospirillum lipoferum 4T has original properties such as nonmotility, melanin synthesis, and laccase activity. Following random Tn5 mutagenesis in A. lipoferum 4T, we obtained 10 mutants which were affected in melanization and laccase activity. The class 1 mutants, with intermediate levels of laccase activity, showed some coloration; the class 2 mutants, which were completely negative for laccase activity, were also colorless. The Tn5 localization on the chromosome or on the cryptic 300-MDa plasmid of A. lipoferum 4T was proven by hybridization for all class 1 mutants or for most class 2 mutants, respectively.