Aims: To assess the efficiency of a Brucella melitensis B115 rough strain, naturally devoid of anticomplementary activity, used as antigen in a complement fixation test (CFT) to detect antibodies induced by Brucella strains with rough phenotype, such as Brucella abortus RB51, Brucella ovis and Brucella canis. Methods and Results: Complement fixation testing was performed on sera from RB51-vaccinated cattle and buffaloes, B. ovis-infected sheep and B. canis-infected dogs using B115, RB51 and the hot saline extract (HSE) as antigens. The B115-based CFT proved highly sensitive and specific in detecting rough antibodies and its efficiency was comparable with that of RB51 and HSE-based CFT. Conclusions: Brucella melitensis B115 can be successfully used as an antigen in CFT to detect antibodies induced by Brucella rough strains. Significance and Impact of the Study: Brucella melitensis B115 antigen may represent an improvement over Brucella rough strains for Brucella antibody detection by CFT, thus enhancing the efficiency of brucellosis surveillance systems. Owing to the absence of anticomplementary activity, it does not require particular growth conditions or modifications and can be accurately standardized. The B115-based CFT may constitute a suitable supplementary test for the diagnosis of human infections owing to rough Brucellae.