The interplay between triggering bacteria and HLA-B27 in the pathogenesis of the spondyloarthropathies remains one of the most active areas of investigation in the rheumatic diseases. This has proved difficult to study systematically in the clinical setting, and in this study we utilized a rat model to address the influence that B27-related immunity may have on the process of generating anti-Chlamydia immunity. When splenocytes from HLA-B27 DNA-immunized Lewis (LEW) animals received restimulation in vitro with Chlamydia-treated cells from B27-transgenic LEW rats, we observed that in addition to the expected CTL recognition of HLA-B27, there was also anti-Chlamydia CTL killing of Chlamydia-sensitized syngeneic fibroblast targets. This was not seen when responding cells in vitro were naive LEW splenocytes. To confirm the existence of CTLs recognizing both HLA-B27 and Chlamydia, LEW rats were immunized with B27-transgenic LEW cells, instead of the B27 DNA construct. Splenocytes from the immune rats were restimulated in vitro with Chlamydia-treated B27-transgenic LEW cells. In this instance, the CTLs retained the allele-specific recognition of HLA-B27, as well as recognition of Chlamydia-sensitized syngeneic fibroblasts. Thus, if there is prior expansion of an immune response against HLA-B27, then the resulting splenocytes demonstrate a reduced threshold for generating a primary anti-Chlamydia CTL response. These studies implicate a dynamic interrelationship between recognition of HLA-B27 and Chlamydia trachomatis. The results may have implications for deciphering the cellular basis of Chlamydia-induced reactive arthritis.