Diagnosis of brucellosis is based on clinical symptoms and positive laboratory results. Because specificity of the serology tests is not satisfactory and sensitivity of the culture is low, polymerase chain reaction (PCR) can be an alternative method in making the final decision in some doubtful cases. In this study, results of standard tube agglutination, specific immunoglobulin (Ig) G and IgM by enzyme-linked immunosorbent assay, and culture were compared with PCR results in 45 patients with brucellosis referred to the Imam Khomeini hospital, Tehran, Iran. The BACTEC (Difco Laboratories, Inc., Sparks, Md) system was applied to isolate the organisms from blood samples, which were then identified by the urease, oxidase, H2S production, and catalase tests. Primer pair was applied to amplify a 223-base-pair fragment of the bcsp31 gene. Analysis of the results revealed that IgG test, IgM test, standard tube agglutination, culture, and PCR results were positive in 97.77%, 40%, 71.11%, 31.11%, and 48.88% of the cases, respectively.