Macrophages are potential candidates for antigen presentation to chlamydial-specific CD4+ T cells. We have studied the kinetics of chlamydial antigen processing and presentation by using paraformaldehyde-fixed bone marrow-derived macrophages (BMDM) and splenic T cells isolated from chlamydia-infected mice. BMDM were inoculated with different multiplicities of heat-killed chlamydial elementary bodies, and at different times postingestion, the macrophages were fixed with paraformaldehyde and used as antigen-presenting cells in T-cell proliferation assays. T-cell proliferative responses were shown to be dependent on the chlamydial inoculum size, with a multiplicity of 10 chlamydiae per macrophage producing optimum T-cell proliferation. Temporal experiments showed that peak T-cell proliferative responses occurred between 4 and 12 h postingestion of chlamydiae by BMDM. T cells proliferated strongly to antigen when presented by H-2-matched BMDM but not when presented by H-2-disparate BMDM, demonstrating that T-cell recognition of processed chlamydial antigen was major histocompatibility complex restricted. BMDM inoculated with 10 chlamydiae per cell and fixed at 8 h postinoculation were shown to be as stimulatory to T cells as conventional splenic antigen-presenting cells. Because large numbers of BMDM can be propagated in vitro, and experimental conditions that provide optimum presentation of processed chlamydial antigen to chlamydia-specific CD4+ T cells can be defined, BMDM may be a potentially useful source for the isolation of naturally processed parasite antigen from major histocompatibility complex class II molecules.