To aid in the development of novel efficacious vaccines against brucellosis, Omp25 was examined as a potential candidate. To determine the role of Omp25 in virulence, mutants were created with Brucella abortus (BA25), Brucella melitensis (BM25), and Brucella ovis (BO25) which contain disruptions in the omp25 gene (Deltaomp25 mutants). Western immunoblot analysis and PCR verified that the Omp25 protein was not expressed and that the omp25 gene was disrupted in each strain. BALB/c mice infected with B. abortus BA25 or B. melitensis BM25 showed a significant decrease in mean CFU/spleen at 18 and 4 weeks post-infection, respectively, when compared to the virulent parental strain (P<0.05, n=5). Mice infected with B. ovis BO25 had significantly lower mean CFU/spleen counts from 1 to 8 weeks post-infection, at which point the mutant was cleared from the spleens (P<0.01, n=5). Murine vaccination with either BM25 or the current caprine vaccine B. melitensis strain Rev. 1 resulted in more than a 2log(10) reduction in bacterial load following challenge with virulent B. melitensis (P<0.01, n=5). Vaccination of mice with the B. ovis mutant resulted in clearance of the challenge strain and provided 2.5log(10) greater protection against virulent B. ovis than vaccine strain Rev. 1. Based on these data, the B. melitensis and B. ovis Deltaomp25 mutants are interesting vaccine candidates that are currently under study in our laboratory for their safety and efficacy in small ruminants.