In patients with Lyme neuroborreliosis, inflammation and symptoms of fatigue and malaise occur out of proportion to the relatively low number of spirochetes present. Previous studies have identified interleukin-6 (IL-6) as a candidate molecule for amplification of CNS inflammation in this disease. We pursued this possibility by measuring cytokine gene expression by reverse-transcriptase polymerase chain reaction (RT-PCR) in the brain of rhesus macaques actively infected with Borrelia burgdorferi. Samples of brain tissue were screened for IL-6 and interferon gamma using RT-PCR-ELISA, a technique that uses RT-PCR, subsequent hybridization of the PCR product with a biotinylated probe, and capture and ELISA readout of hybridization product. The number of copies in positive samples was then quantitated using qRT-PCR-ELISA, in which wild-type cytokine cDNA competes with recombinant competitor DNA in the PCR. Elevated levels of IL-6 cDNA and, to a lesser extent, interferon gamma were detected in three of three nonhuman primates with persistent infection with B burgdorferi, whereas the brains of three uninfected animals and undetectable levels of gene expression of these cytokines. These data support the hypothesis that cytokines such as IL-6 are important amplification molecules for CNS inflammation in Lyme neuroborreliosis.