The specificity of the human T cell response to Chlamydia trachomatis was investigated by stimulating lymphocytes from 16 case patients with urogenital infection by use of a size-fractionated serovar D lysate. Considerable heterogeneity was found among case patients, and multiple protein fractions were recognized in each specimen. Mass spectrometry analysis of the 30-42-kDa T cell-stimulating region identified 10 C. trachomatis proteins. Of these, CT583, CT603, and CT610 were identified as strong antigens that induced significantly higher levels of IFN- gamma secretion in PBMCs from case patients, compared with PBMCs from control donors. All 3 proteins were recognized in specimens from case patients infected with serovars D-F, the most prevalent serovars. McDonald-Kreitman and Tajima's D tests involving clinical isolates from the same samples showed evidence for frequency-dependent selection on ct583. We predict that CT583 is a target of acquired protective immune responses in humans.