Fundamental understanding of rabbit immunology and the use of the rabbit as a disease model have long been hindered by the lack of immunological assays specific to this species. In the present study, we sought to develop a method to quantitate cytokine expression in rabbit cells and tissues. We report the development of a quantitative real-time RT-PCR method for measuring the relative levels of rabbit IFN-gamma, IL-2, IL-4, IL-10 and TNF-alpha mRNA. Quantitation was accomplished by comparison to a standard curve generated using plasmid DNA containing partial sequences of the relevant cytokines. Experimental studies demonstrate applicability of this assay to quantitate cytokine mRNA levels from rabbit spleen cells following mitogen stimulation. We have further utilized this assay to also examine cytokine expression in rabbit tissues during experimental syphilis infection.