A simple polymerase chain reaction-enzyme immunoassay (PCR-EIA) was employed for the rapid laboratory diagnosis of human brucellosis directly from peripheral blood. Whole blood and serum specimens were collected from 243 patients with acute brucellosis as determined by blood culture, serological tests, and the patients' clinical characteristics and from a control group of 50 healthy individuals. Diagnosis of brucellosis was established in 179 cases by isolation of Brucella spp. in blood culture and in 64 cases by clinical signs and serological investigation. Following the amplification of a 223-bp sequence of a gene that codes for the synthesis of an immunogenic membrane protein specific for the Brucella genus, the amplified product was detected in a microtiter plate by hybridization. Two hundred forty-one of the 243 patients tested had detectable Brucella DNA in either whole blood or serum specimens: 149 (61.3%) patients were positive in both whole blood and serum specimens, 43 (17.7%) were positive in serum specimens only, and 49 (20.2%) were positive in whole blood specimens only. The diagnostic specificity of the PCR-EIA assay for both specimen categories was 100%, while the sensitivity was 81.5% for whole blood specimens, 79% for serum specimens, and 99.2% for whole blood and serum specimens combined. The results suggest that the detection of Brucella DNA in whole blood and serum specimens by PCR-EIA assay is a sensitive and specific method that could assist the rapid and accurate diagnosis of acute human brucellosis.