A polymerase chain reaction (PCR) assay was designed to amplify DNA fromRochalimaea henselae, Rochalimaea quintanaandAfipia felisin the purulent material from lymph nodes in three patients with clinical cat-scratch disease (CSD) and two patients with lymphadenitis from other causes. All of the patients with CSD had positive immunofluorescent antibody serology forR. henselae, while none of the controls was positive. PCR amplification confirmed the presence ofR. henselaeDNA and the absence ofR. quintanaand A.felisDNA in the purulent material from CSD patients. PCR samples from control patients were negative. The PCR amplification ofR. henselaeDNA was performed quickly and with great sensitivity and specificity. It confirmed the presence ofR. henselaein the CSD patients and eliminated the need for more extensive diagnostic and therapeutic procedures.