Aims: To compare the culture and PCR methods for detection of Brucella melitensis in blood and lymphoid tissue samples obtained from slaughtered sheep (n = 162) testing positive/negative in serological tests (Rose Bengal test and serum agglutination test). Methods and Results: Of 162 sheep examined, 45 were positive and 117 negative in serological tests. A PCR assay based on a pair of Br. melitensis-specific primers was used to detect DNA in blood and lymphoid tissue. Brucella melitensis was isolated from 1.2% (2/162) and 17.2% (28/162) of the blood and lymphoid tissue samples respectively. Positive PCR products with a molecular size of 731 bp were obtained from 27.7% (45/162) of blood and 29.0% (47/162) of lymphoid tissue samples. Conclusions: The species-specific PCR assay detected a higher number of Br. melitensis DNA both from serologically positive (P < 0.01 in blood PCR, P < 0.001 in tissue PCR) and serologically negative (P < 0.001 in both blood PCR and tissue PCR) sheep compared with classical bacteriological culture methods. Significance and Impact of the Study: The results emphasize the importance of using more than one type of diagnostic technique for the detection of animals positive for brucellosis, especially with epidemiological purposes. © 2007 The Authors.