ABSTRACT Chlamydia species represent a paradigm for understanding successful obligate intracellular parasitism. While C. trachomatis and C. pneumoniae are both prevalent human pathogens, C. pneumoniae respiratory infections are likely most common. Acute C. pneumoniae infections manifest as community acquired pneumonia, bronchitis, and sinusitis while additional inflammatory sequelae are associated with chronic infection. Advances in genetic tractability have facilitated considerable progress in characterizing C. trachomatis pathogenesis. Unfortunately, similar progress has lagged for C. pneumoniae, leading to a paucity in details regarding molecular mechanisms of infection and precluding informative approaches leveraging comparative studies. To overcome this barrier, we will engineer plasmid systems enabling allelic replacement and ectopic gene expression for C. pneumoniae. These new technologies will be applied in proof-of-principle studies to address functional aspects manifested by a pair of divergent type III secreted effectors employed by C. pneumoniae to manipulate host cell biology. At the end of these studies, we will have established new approaches that will benefit the entire Chlamydia research community and advance the understanding of how chlamydial species have evolved to accomplish common developmental requirements.