Cat scratch disease (CSD) is mostly due to Bartonella henselae after inoculation of the organism through a skin injury. Since the causative bacteria cannot be easily cultured from human lymph node samples, the diagnosis usually relies on epidemiological, clinical, histological, and serological criteria (classical criteria). A study was performed to determine the diagnostic value of PCR analysis for the detection of B. henselae for the diagnosis of CSD and its place in the diagnostic strategy alongside the classical criteria. Over a 7-year period, lymph node biopsy specimens or cytopunctures from 70 patients were systematically tested by PCR for the presence of B. henselae DNA (htrA gene) in the Bacteriology Laboratory of the Hôpitaux Universitaires de Strasbourg. Serological testing by an immunofluorescence assay for B. henselae antibodies was also performed for each patient, and clinical, epidemiological, and histological data were collected. The patients were then divided into two groups according to the number of positive diagnostic criteria for CSD: 29 patients with definite CSD (two or more classical criteria) and 15 patients with possible CSD (less than two classical criteria). The remaining 26 patients for whom another diagnosis was retained were used as a control group. Among all criteria, PCR analysis had the best specificity (100%). The PCR assay for B. henselae was positive for 22 (76%; 95% confidence interval [CI95], 56.5 to 89.7%) of the 29 definite CSD patients and 3 (20%; CI95, 4.3 to 48.1%) of the 15 possible CSD patients. We then studied combinations of diagnostic criteria, including B. henselae PCR analysis. The best diagnostic performance was observed if at least two criteria were present among serologic, epidemiologic, histological, and molecular criteria.