Bacillus clausii SIN is one of the four strains of B. clausii composing a probiotic administered to humans for the prevention of gastrointestinal side effects due to oral antibiotic therapy. The strain is resistant to kanamycin, tobramycin, and amikacin. A gene conferring aminoglycoside resistance was cloned into Escherichia coli and sequenced. The gene, called aadD2, encoding a putative 246-amino acid protein, shared 47% identity with ant(4')-Ia from Staphylococcus aureus, which encodes an aminoglycoside 4'-O-nucleotidyltransferase. Phosphocellulose paper-binding assays indicated that the gene product was responsible for nucleotidylation of kanamycin, tobramycin, and amikacin. The aadD2 gene was detected by DNA-DNA hybridization in the three other strains of the probiotic mixture and in the reference strain B. clausii DSM8716, although it did not confer resistance in these strains. Mutations in the sequence of the putative promoter for aadD2 from B. clausii SIN resulted in higher identity with consensus promoter sequences and may account for aminoglycoside resistance in that strain. The aadD2 gene was chromosomally located in all strains and was not transferable by conjugation. These data indicate that chromosomal aadD2 is specific to B. clausii.