The brucellae are Gram-negative pathogens that cause brucellosis, a zoonosis of worldwide importance. The genus Brucella includes smooth and rough species that differ in that they carry smooth and rough lipopolysaccharides, respectively. Brucella abortus, B. melitensis, and B. suis are typical smooth species. However, these smooth brucellae dissociate into rough mutants devoid of the lipopolysaccharide O-polysaccharide, a major antigen and a virulence determinant encoded in regions wbo (included in genomic island-2) and wbk. We demonstrate here the occurrence of spontaneous recombination events in those three Brucella species leading to the deletion of a 5.5-kb fragment carrying the wbkA glycosyltranferase gene and to the appearance of rough mutants. Analysis of the recombination intermediates suggested homologous recombination between the ISBm1 insertion sequences flanking wbkA as the mechanism generating the deletion. Excision of wbkA was reduced but not abrogated in a recA-deficient mutant, showing the existence of both RecA-dependent and -independent processes. Although the involvement of the ISBm1 copies flanking wbkA suggested a transpositional event, the predicted transpositional joint could not be detected. This absence of detectable transposition was consistent with the presence of polymorphism in the inverted repeats of one of the ISBm1 copies. The spontaneous excision of wbkA represents a novel dissociation mechanism of smooth brucellae that adds to the previously described excision of genomic island-2. This ISBm1-mediated wbkA excision and the different %GC levels of the excised fragment and of other wbk genes suggest that the Brucella wbk locus is the result of at least two horizontal acquisition events.