The antigenically variant Chlamydia trachomatis major outer membrane protein (MOMP) is a target of antibody-mediated neutralization in vitro, and it is an important protein for designing a subunit vaccine. Knowledge of MOMP T-cell determinants will be essential to elicit rapid and strong immune responses following an encounter with infectious organisms. C. trachomatis-specific T-cell lines were derived from MOMP-immunized BALB/c mice and selected with intact organisms. We used these short-term T-cell lines to identify determinants of MOMP that could be recognized by T cells following processing of the intact organism. T-cell line proliferation in response to overlapping MOMP peptides showed that only a peptide encompassing the third variable segment (VS3) elicited a strong proliferative response. We further mapped determinants within the VS3 peptide and found that a sequence-conserved portion of the VS3 peptide elicited T-cell proliferation of T-cell lines from BALB/c mice. Thus, unlike the response to several MOMP peptides with unselected T cells, development of short-term T-cell lines with intact organisms restricted the repertoire of antigens capable of being recognized by MOMP-specific T cells.