A single-tube nested PCR assay identifying a 52 bp fragment from the genus-specific Brucella IS711 gene was used prospectively in clinical practice for the diagnosis of human brucellosis in Kuwait. Patients with suspected brucellosis and with other infections were investigated as clinically indicated but in all of them culture, serology and PCR for Brucella were carried out. Out of 263 suspected cases of brucellosis, diagnostic tests were positive in 199, serology was positive in 199 and culture in 89, while the Brucella PCR was positive in 193 (sensitivity 96.98%, 95% confidence interval 94.5-99.5%). Chronic brucellosis, involving symptoms for more than 1 year, was diagnosed in 49 out of these 193 patients. Diagnoses in four out of the six patients with positive serology but negative PCR and culture were non-Brucella bacterial meningitis and viral meningitis. False-negative PCR results were possible in the remaining two; both had been on long-term antibiotics for previously diagnosed brucellosis but their adherence may have been questionable, allowing a relapse. The PCR was negative in 244 patients with other infections (specificity 100%) and in 180 control subjects with negative Brucella culture and serology. PCR results were available within 24 h of sample receipt, providing diagnostic information rapidly. The PCR is expensive and technically demanding and may not be appropriate for all cases. Future studies will help define how it may best be used. For example Brucella and tuberculous meningitides can be very similar, Brucella PCR may allow prompt distinction between the two, avoiding the need for prolonged empirical treatment for both.