Brucella melitensis strains may occur as either smooth or rough variants depending on the expression of O-polysaccharides (OPS) as a component of the bacterial outer membrane lipopolysaccharide (LPS). The wboA gene, which codes for the enzyme glycosyl transferase, is essential for the assembly of O-chain in Brucella. Deletion of wboA in smooth virulent B. melitensis 16M results in a rough mutant designated WRR51. We developed a flow cytometric method to determine the proportion of B. melitensis cells displaying surface O-polysaccharide (OPS) in liquid culture. OPS was detected using polyclonal antibodies from rabbits immunized with smooth (S) or rough (R) Brucella LPS. First, we evaluated the binding of these antibodies to 16M (S), WRR51 (R) and complemented WRR51 expressing the wboA gene (S) as well as to their corresponding GFP-expressing derivative strains 16M/GFP, WRR51/GFP and WRR51/GFP+wboA. The rough mutants did not react with anti-S-LPS nor did the smooth strains react with anti-R-LPS. Second, using different ratios of 16M/GFP and WRR51/GFP, we were able to detect the presence of 1% rough bacteria spiked into a sample of smooth organisms. Third, we evaluated the purity of cultures of B. melitensis strains grown in a fermenter. These flow cytometric methods may be useful for quality control of process development for large-scale vaccine production.