Brucellosis is one of the most common zoonotic diseases in Iran and human brucellosis is endemic in all parts of the country. Growth of Brucella is slow and blood culture of these bacteria by use of classical methods is time-consuming. Furthermore, in endemic area culture is required for definitive diagnosis. In the present study, direct urease test and acridine orange staining were tried on the BACTEC blood culture broths for early presumptive identification of Brucella growth. Blood cultures were attempted in 102 seropositive patients. In the forty one blood cultures positive for Brucella, coccobacilli were seen in broth smears stained with acridine orange stain, and also were urease test positive, thus providing presumptive identification of Brucella growth. Urease test was negative and bacteria were not seen in the broth smears of the remaining 61 broths negative for Brucella growth. Because of simplicity, reliability and reproducibility, these tests can be routinely incorporated in the laboratory for diagnosis of brucellosis.