One of the properties of bacteria is their capacity to acquire large fragments of genomic DNA from other bacteria or to loose important parts of their own genome. Such fragments include genomic islands (GIs); nine GIs are present in Brucella, including genomic island 3 (GI-3), present in B. abortus, B. melitensis and B. ovis. The GI-3 have 29 open reading frames (ORFs) most of them with unknown function. Within the GI-3, the ORFs BAB1_0267 encodes a hypothetical protein sharing a SH3 domain and BAB1_270 a zinc-dependent metallopeptidase. We have obtained deletion mutants for BAB1_0267 and BAB1_0270 ORFs present within GI-3, which have been named the Delta 0267 and Delta 0270, respectively; in both cases the mutation did not affect the growth of bacteria. Both mutants were evaluated with respect to their growth rates, their ability to invade and replicate in the non-professional and professional phagocytes, HeLa and J774.A1 cells, respectively. Their persistence in the spleens of mice was also evaluated. The mutants efficiently invaded HeLa and J774.A1 cells but both mutants showed a decreased intracellular survival in macrophages and HeLa cells 72 and 96 h post-infection, respectively, and were non-detected in J774.A1 cells 120 h post infection. With respect to in vivo persistence Delta 0267 was detected through the fourth week while Delta 0270 decreased at 7 days disappearing the second week. Our results indicated that deletion of BAB1_0267 and BAB1_270 are necessary to establish an optimal infectious process in B. abortus 2308, having more effect the deletion of ORF BAB1_0270. Therefore these ORFs, principally BAB1_0270 are important virulent of B. abortus. (C) 2014 Elsevier B.V. All rights reserved.