The obligate intracellular bacterium Chlamydia trachomatis is associated with human diseases ranging from blinding trachoma to sexually acquired genital infections and the systemic disease lymphogranuloma venereum (LGV). We have previously reported the isolation and culture of protective murine cytotoxic T lymphocytes (CTL) following intraperitoneal infection with C. trachomatis serovar L2, a serotype associated with human LGV. In this report, we now demonstrate that CTL can also be primed following introduction of C. trachomatis serovar L2 into the uterus or ovarian bursa of mice. We also describe Chlamydia-specific CTL lines isolated following murine infection with a typical human urogenital isolate of C. trachomatis (serovar D) and show that such CTL can be primed by intraperitoneal, intrauterine, or intrabursal infection. Last, we demonstrate that these murine CTL lines respond to multiple serovars, recognizing and lysing cells infected with C. trachomatis serovars B, C, D, F, J, K, L2, and L3, representative of organisms causing blinding trachoma, genital infection, and LGV.