Human brucellosis is a significant public health problem in many Mediterranean countries including Greece. The conventional serological methods, as well as blood cultures, have serious limitations, especially in chronic, relapsing, and focal forms of the disease. Four different PCR assays were applied in 4,926 buffy coat, whole-blood, and serum samples received from 200 patients admitted with brucellosis to the Infectious Diseases Hospital, Thessaloniki, Greece, for the rapid diagnosis of acute infection and relapses and compared to blood culture and serological tests (i.e., Wright's seroagglutination test, Coombs' antibrucella test, and the complement fixation test). The four PCR assays had excellent sensitivity and specificity and were able to detect all of the cases of acute disease. Buffy coat and whole blood were the optimal specimens. All four PCR assays were negative in all follow-up samples from 183 patients who had completed a successful treatment and were positive in all follow-up samples from 17 patients who had relapses in the first year after therapy, including the times of the relapses. In conclusion, PCR is a very useful tool for the rapid diagnosis of acute brucellosis and a good marker for the posttreatment follow-up and the early detection of relapses.