Diagnosis of brucellosis can be difficult in certain scenarios where conventional microbiological techniques have important limitations. The aim of this study was to develop a LightCycler Quantitative PCR assay in serum samples to discriminate between active and past brucellosis. In total, 110 serum samples from 46 brucellosis patients and 64 controls, including persons who had recently been treated for brucellosis, asymptomatic persons exposed to brucellosis, and patients with febrile syndromes involving a differential diagnosis with brucellosis, were studied. Brucella spp.-specific sequences of the PCR primers and probe were selected from the gene encoding an immunogenic membrane protein of 31 kDa (BCSP31). The analytical sensitivity was 1 x 10(1) fg of Brucella DNA. The mean threshold cycles for brucellosis patients and controls were 31.8 +/- 1.7 and 35.4 +/- 1.1, respectively (p <0.001). The best cut-off for bacterial DNA load was 5 x 10(3) copies/mL. At this cut-off, the area under the receiver operating characteristic curves was 0.963 (95% CI 0.920-1.005), with a sensitivity of 93.5% and a specificity of 98.4%. Under the assay conditions, the LightCycler Quantitative PCR in serum samples seems to be highly reproducible, rapid, sensitive and specific. It is therefore a useful method for both the initial diagnosis and the differentiation between past and active brucellosis.